CN108828209A - The preparation method of microballoon for immune detection - Google Patents
The preparation method of microballoon for immune detection Download PDFInfo
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- CN108828209A CN108828209A CN201810694550.6A CN201810694550A CN108828209A CN 108828209 A CN108828209 A CN 108828209A CN 201810694550 A CN201810694550 A CN 201810694550A CN 108828209 A CN108828209 A CN 108828209A
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- 241000220324 Pyrus Species 0.000 description 1
- YHSHAHLOHLZILM-UHFFFAOYSA-L S(=S)(=O)([O-])[O-].[Na+].C(C)[Hg].[Na+] Chemical compound S(=S)(=O)([O-])[O-].[Na+].C(C)[Hg].[Na+] YHSHAHLOHLZILM-UHFFFAOYSA-L 0.000 description 1
- 102000054727 Serum Amyloid A Human genes 0.000 description 1
- 108700028909 Serum Amyloid A Proteins 0.000 description 1
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 1
- 239000007997 Tricine buffer Substances 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 239000007998 bicine buffer Substances 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- HHKZCCWKTZRCCL-UHFFFAOYSA-N bis-tris propane Chemical compound OCC(CO)(CO)NCCCNC(CO)(CO)CO HHKZCCWKTZRCCL-UHFFFAOYSA-N 0.000 description 1
- 238000011094 buffer selection Methods 0.000 description 1
- 235000010331 calcium propionate Nutrition 0.000 description 1
- 239000004330 calcium propionate Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 238000002484 cyclic voltammetry Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 108700003601 dimethylglycine Proteins 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000010228 ethyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004403 ethyl p-hydroxybenzoate Substances 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 102000045903 human LPA Human genes 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000013016 learning Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 235000021017 pears Nutrition 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 239000000249 polyoxyethylene sorbitan monopalmitate Substances 0.000 description 1
- 235000010483 polyoxyethylene sorbitan monopalmitate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940101027 polysorbate 40 Drugs 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229960003471 retinol Drugs 0.000 description 1
- 235000020944 retinol Nutrition 0.000 description 1
- 239000011607 retinol Substances 0.000 description 1
- 238000010850 salt effect Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of preparation method of microballoon for immune detection, this method includes 1) diluting the microballoon and object to be marked using buffer；2) mixed solution of the microballoon Yu object to be marked is obtained；3) crosslinking agent is added to be incubated for；4) blocking agent is added；Wherein, the microballoon is carboxyl microballoon；The pH of buffer in step 1) is 6.3~9.5.The invention further relates to the microballoon being prepared by this method and contain the kit of the microballoon.
Description
Technical field
The invention belongs to field of immunodetection, and in particular to a method of prepare the carboxyl microballoon for immune detection.
Background technique
Immunological detection method mainly has enzyme immunoassay, immunoturbidimetry, colloid gold immune measuring method, chemiluminescence
Measuring method, fluorescence immunoassay, radiommunoassay etc..Wherein, the diagnostic reagent prepared using the immunological method of microballoon,
By that can be utilized by automatic analytical instrument, it is widely used in clinical examination etc. in recent years.It is used for by microballoon
When immune detection diagnoses, microballoon is marked in the usually used object to be marked in conjunction with measure object.In the sample containing survey
When determining object, by making it in conjunction with the object to be marked that microballoon is loaded, microballoon is caused to be aggregated.To by measuring the agglutination
Degree determines the presence or absence of measure object or its content.
Currently, microballoon and object connection method to be marked mainly have physical absorption and chemical coupling.Wherein, chemical coupling is benefit
Object to be marked and microballoon are linked together by chemical reaction with activatable crosslinking agent, the stability after connection is more compared with physical absorption
Good (JL Ortega-Vinuesa et al., Journal of Colloid&Interface Science, 1995).For changing
The microsphere surface for learning coupling can be modified with some chemical groups, as carboxyl, amino and hydroxyl (it is micro- to correspond to carboxyl microballoon, amino
Ball and hydroxyl microballoon) etc..And among those, carboxyl microballoon application is more extensive.
It is well-known using the method that activator prepares carboxyl microballoon, for example CN106353507A discloses a kind of incite somebody to action
Method of the antibody coupling to microballoon, this method addition EDC and sulfo-NHS priming reaction 15 after particle is resuspended with buffer
Minute, eccentric cleaning and ultrasound resuspension after reaction；The antibody of dissolution is mixed with particle then and carries out coupling reaction again, after coupling
Eccentric cleaning and resuspension need to be carried out again.For another example CN105606822A discloses a kind of use antibody sensitized polystyrene latex
The method of particle, it is specific to wash latex three times and ultrasonic disperse with the MES of pH5.0, EDAC is added and activates 15 minutes, washs 2 times
And ultrasonic disperse, antibody mixing is then sequentially added, closing obtains sensitization particle.
However, the above method is not only centrifuged during the preparation process to remove free activator and not tie with free
The antibody of conjunction, it is also necessary to ultrasonic resuspension be carried out to microballoon so that it disperses.Although these steps eliminate extra component and solve
Agglutinophilic problem when the preparation of carboxyl microballoon, but make the operating process to become low efficiency and time-consuming, it is unfavorable for preparation of industrialization.
In response to this, it has been proposed that the method for being not required to purifying, such as such side is disclosed in CN107167586A
Method：Latex is first added to buffer, activation 15 minutes is carried out EDC is added, adjusts pH to 7.60 later, add SLO progress
Cross-linking reaction 2h, is eventually adding the method that two kinds of blocking agents obtain finished product, and this method does not need to carry out centrifugation or ultrafiltration step
Suddenly, to improve preparation efficiency.
But this method also needs additionally to adjust pH value later there is still a need for first microballoon is activated for a period of time with crosslinking agent
It just can be carried out cross-linking reaction afterwards, this inevitably extends preparation time, increases the complexity of operation to be not easy in work
It is applied in industry.
Therefore, in field of immunodetection, however it remains shorter carboxyl microballoon labeling method simpler to operation, time-consuming
Demand.
Summary of the invention
For this purpose, the technical problem to be solved in the present invention is to provide a kind of carboxyl microballoon labeling methods easy to operate.It is another
Aspect further solves the problems, such as how to prevent microballoon to be aggregated in carboxyl microballoon labeling process.Another further aspect also solve how
The problem of improving the labeling effciency of carboxyl microballoon.Another aspect also solves the problem of stability for improving carboxyl microballoon.
In order to achieve the above objectives, the present inventor studies the labeling method of carboxyl microballoon, and is surprised to find that pass through
The pH of adjustment microballoon dilution buffer and antibody dilution buffer can be added after crosslinking agent (i.e. for priming reaction),
PH is still within the scope of most suitable, and carboxyl microballoon has better stability after this ensures sensitization, without carrying out to microballoon
It activates in advance, without carrying out additional adjustment to pH after addition of the cross-linking agent；In the course of the research, inventor be also found to
It is few by reduce the step for crosslinking agent is preactivated (first microballoon is contacted with object to be marked mix add crosslinking agent make it is micro-
Ball and object to be marked are crosslinked), microballoon sensibility is reduced, makes to be not easy to agglomerate in labeling process, thus not necessarily in mark
Centrifugation step and/or dispersion steps are used during note.
That is, adjusting while by pH parameter and markers step, for the label of carboxyl microballoon, the present invention is real
Show simplified operating process and shortened the effect of operating time, while not needed large centrifugal equipment in production, improves production
Safety.And as follows confirmed, the labeled microballoon that this method is prepared can be used among immune detection.
Accordingly, it the present invention provides a kind of method for marking microballoon, the described method comprises the following steps：
1) microballoon and object to be marked are diluted using buffer；
2) mixed solution of the microballoon Yu object to be marked is obtained；
3) crosslinking agent is added to be incubated for；
4) blocking agent is added；
5) the carboxyl microballoon after being marked；
Wherein, the microballoon is carboxyl microballoon, and the pH of the buffer in step 1) is about 6.3~9.5.
In some embodiments, the pH of the buffer in step 1) is about 7.0~9.0.
In some embodiments, the dilution in step 1) can be carried out according to any conventional dilution mode, such as can be with
Microballoon and object to be marked are diluted respectively, then mix the two；Or first with buffer dilute microballoon, be then added thereto to
Marker.It in some embodiments, may include the step of diluting microballoon between step 4) and step 5).It preferably, can be with
Microballoon is diluted with buffer commonly used in the art, the buffer includes but is not limited to Good ' s buffer, amino acids buffering
Liquid etc..Under the conditions of the pH involved in the method for the present invention, inventor is further discovered that：To buffer type and its ion buffer
Selection, can help to the agglutination for further preventing microballoon and improve labeling effciency.
Therefore, method of the invention further relates to：
In one embodiment, the buffer in step 1) is Good ' the s buffering that ionic strength is about 3~30mM
Liquid.
Preferably, Good ' the s buffer is selected from one of HEPES, MPOS, MES, TES, TAPS and HEPPSO
Or more.
Preferably, the ionic strength of Good ' the s buffer is about 5mmol/L~25mmol/L.
In another embodiment, the buffer in step 1) is the amino acids that ionic strength is about 10~500mM
Buffer.
Preferably, the amino acids buffer is neutral fat race buffered with amino acid liquid and/or basic amine group acid buffering
Liquid.
It is further preferred that the neutral fat race buffered with amino acid liquid is that glycine buffer and/or alanine buffer
Liquid.
It is further preferred that the basic amine group acid buffer is Arginine buffer or lysis buffer.
In some embodiments, the crosslinking agent is EDC, DCC or DIC.
Preferably, the ionic strength of the amino acids buffer is about 15mmol/L~300mmol/L.
In addition, for method of the invention, the present inventors have additionally discovered that：By adjusting the usage amount of crosslinking agent, can make to be lived
The microballoon quantity of change maintains a relatively suitable section, thus help avoid because vie each other after microballoon excess activation to
Marker causes labeling process to be aggregated.
Therefore, method of the invention further relates to：
In one embodiment, the weight ratio of crosslinking agent and microballoon is about 0.5%~4.0%.
Preferably, the weight ratio of crosslinking agent and microballoon is about 1.0%~3.5%.
In addition to the above method and further embodiment, method of the invention is further related to：
In one embodiment, the object to be marked is amino-containing object to be marked.
Preferably, the amino-containing object to be marked is selected from one of protein, peptide and polypeptide or more.
It is highly preferred that the amino-containing object to be marked is antigen and/or antibody.
It is further preferred that the antibody is monoclonal antibody and/or polyclonal antibody.
In one embodiment, method of the invention further includes：
The step of addition protective agent, preservative and/or surfactant between step 4) and step 5).
In one embodiment, method of the invention does not include any purification step.
Preferably, the purification step is carried out using ultrafiltration and/or centrifugation.
In one embodiment, method of the invention does not include the steps that any for dispersion microsphere.
Preferably, described the step of being used for dispersion microsphere, is carried out using ultrasound.
On the other hand, the invention further relates to labeled microspheres solutions, are prepared by method of the invention.
It yet still another aspect, the kit includes the present invention the invention further relates to a kind of kit for immune detection
Labeled microspheres solution.
Therefore, method of the invention simplifies the operating process of microballoon labeling method and reduces operation duration, improves life
The safety of production process is more advantageous to industrialization.Further, method of the invention prevents the solidifying of microballoon in labeling process
Collection.Still further, method of the invention also improves the labeling effciency of microballoon.
Detailed description of the invention
Fig. 1 is the appearance picture of the microballoon in labeling process；
Fig. 2 is the appearance picture of the microballoon in labeling process；
It is bent that Fig. 3 shows the dose response of microballoon that experimental group, comparative example and conventional method of the present invention are marked when detecting
Line chart.
Specific embodiment
Below in conjunction with specific embodiment and embodiment, it is specifically described the present invention, advantages of the present invention and various effects
It thus will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating
The present invention is not intended to limit the present invention.
Throughout the specification, unless otherwise specified, terms used herein are interpreted as usual in this field
Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has leads with belonging to the present invention
The identical meaning of the general understanding of field technique personnel.Contradiction if it exists, this specification are preferential.
Microballoon
In the present invention, " microballoon " refers to the carrier that can be used as object to be marked with the small entity for immunology detection
Set, diameter are generally less than 5 microns.
As used in the present invention, " carboxyl microballoon " refers to that surface modification has and covalently ties for the aminoterminal with object to be marked
The microballoon of the carboxyl of conjunction.There is a bridging chemistry arm between carboxyl microballoon and antibody, reduces steric effect, not only increase anti-
The Percentage bound of body, but also suitable three-dimensional space stereochemical structure is provided for antibody, it is effectively protected antibody and antigen binding
Active region.Foreign countries have more companies and provide the microsphere particles raw material that surface modification has carboxyl, for example, Sichuan new material is public
Take charge of the carboxyl microballoon of production, the carboxyl microballoon etc. of Bangs Laboratories company production.
In the present invention, the carboxyl microballoon of any suitable type can be used, if its can with contain the to be marked of amino
Object coupling.Common microballoon may be, for example,：Latex (such as it is polystyrene microsphere, chitosan microball, polylactic acid microsphere, poly-
Acrylic microspheres and/spherex etc.), fluorescent microsphere, magnetic bead.However, the present invention is not limited thereto.
For the diameter of carboxyl microballoon, the present invention is not particularly limited, such as the diameter within the scope of 20nm~5000nm
It can be used in labeling method of the invention.
Object to be marked
As used in the present invention, " object to be marked ", which refers to, can be coupled with carboxyl microballoon and can be used to specifically bind sample
Present in determinand substance.
It is well known in the art with the substance of determinand specific binding, for example, in a kind of diagnosis of hepatitis b
Method in, hepatitis B surface antibody can be used as determinand, and hepatitis B surface antibody can be used as this hair in the case
The object to be marked of bright microballoon.Similarly, in these known objects to be marked, which object to be marked as known to those skilled in the art
It can be coupled with carboxyl microballoon.
For the type of object to be marked, the present invention is not particularly limited, as long as it can be coupled with carboxyl microballoon.Example
Such as, object to be marked can be antibody or antigen.For another example, object to be marked can be polypeptide or fluorescent chemicals
When object to be marked is antibody, monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody can be selected from
Or antibody fragment (preferably with the segment of antigen binding) etc..
In some embodiments, antibody sources can be rabbit source, source of mouse, goat source, sheep source, Ji Yuan and/or source of people
Deng.
When object to be marked is antigen, antigen or its segment (preferred antigens epitope) etc. can be selected from.
Object to be marked of the invention can be obtained by purifying, expression and/or the modes such as artificial synthesized.
Can include for illustrative object to be marked：Antiserum amyloid A antibody, RBP ELISA antibody, β
2 microglobulin polyclonal antibodies, anti-human cystatin C polyclonal antibody, rabbit-anti human lipoprotein a antibody, anti-myoglobins are polyclonal
Antibody or anti-human d-dimer monoclonal antibody etc..However, the present invention is not limited thereto.
In some embodiments, the object to be marked can be modified.
Buffer
It include microballoon dilution buffer and antibody dilution buffer in the method for the present invention.
Microballoon dilution buffer and antibody dilution buffer can be selected from as Good ' s buffer or buffered with amino acid liquid.
The pH of buffer of the invention can for 6.3~9.5, for example, 6.4,6.5,6.6,6.7,6.8,6.9,7.0,
7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、 8.5、8.6、8.7、8.8、8.9、
9.0,9.1,9.2,9.3,9.4 or 9.5.
Inventor is further discovered that：In the methods of the invention, Good ' the s buffer of low ionic strength can further drop
The risk of low latex agglutination.
Therefore, microballoon dilution buffer and antibody dilution buffer of the invention is in one preferred embodiment
Good ' the s buffer that ionic strength is about 3~30mM.
Good ' s buffer is also known as zwitterionic buffer, they are when as buffer system with common characteristic：
1) pKa is between 6-8；2) solubility in water is high；3) it is not easy to penetrate biomembrane；4) salt effect is small；5) ion concentration,
The influence of solution composition and temperature to dissociation is small；6) compound or precipitating are not generated with metal ion；7) buffer chemical is steady
It is fixed；8) light absorption is small in Uv and visible light wave-length coverage；9) salt of easily obtained high-purity.Common Good ' s buffer packet
It includes：MES,Bis-Tris, ADA,ACES,PIPES,MOPSO,Bis-Tris Propane,BES,MOPS,HEPES,TES,
DIPSO、TAPSO、TRIZAMA、HEPPSO、POPSO、EPPS、TEA、Tricine、Bicine、 TAPS、AMPSO、CHES、
CAPSO or AMP etc..
In preferred embodiment, Good ' s buffer of the invention can be MOPS, HPEPS, MES, TES, TAPS
With one of HEPPSO or more.
When microballoon dilution buffer and antibody dilution buffer are selected from Good ' s buffer, ionic strength can be
3mM, 5mM, 10mM, 15mM, 20mM, 25mM, 26mM, 27mM, 28 mM, 29mM or 30mM.
In addition, inventor has been unexpectedly discovered that：In the methods of the invention, the use of buffered with amino acid liquid can further drop
The risk of low latex agglutination, and when using buffered with amino acid liquid, ionic strength can be applicable in a wider scope.It is right
This, it is presumed that slowing down crosslinking rate, to make this is because buffered with amino acid liquid also takes part in crosslinking to a certain extent
Labeling process is less susceptible to be aggregated.
Therefore, in another preferred embodiment, microballoon dilution buffer and antibody dilution buffer are ions
The amino acids buffer that intensity is about 10~500mM.Common buffered with amino acid liquid includes that glycine buffer, alanine are slow
Fliud flushing, valine buffer, leucine buffer, isoleucine buffer, histidine buffering liquid, Arginine buffer or bad ammonia
Acid buffer etc..
In preferred embodiment, amino acids buffer of the invention is neutral fat race buffered with amino acid liquid,
For example, glycine buffer, alanine buffer, valine buffer, leucine buffer or isoleucine buffer.
In another preferred embodiment, amino acids buffer of the invention is basic amine group acid buffer,
Such as Arginine buffer, histidine buffering liquid or lysis buffer.
When microballoon dilution buffer and antibody dilution buffer are selected from amino acids buffer, ionic strength can be with
For 10mM, 15mM, 40mM, 50mM, 100mM, 150mM, 200mM, 250 mM, 300mM, 350mM, 400mM, 450mM or
500mM。
In the method for the invention, microballoon dilution buffer and antibody dilution buffer can be identical or different.One
In a preferred embodiment, microballoon dilution buffer and antibody dilution buffer are identical.
Sealer
As used in the present invention, " sealer " is the closed reagent of carboxylic group for referring to be not associated on microballoon.
It will be appreciated by those skilled in the art that it is any the closed reagent of carboxylic group can be can be used as it is of the invention
Sealer.Illustrative sealer can be selected from protide sealer, such as BSA, casein, skimmed milk power or substance containing amino,
Such as, one or more of TRIS, amino acids sealer (such as alanine, arginine, lysine).
For the dosage of sealer, those skilled in the art can select according to its type, for example, using
The concentration of such as 1~5g/L can be used when BSA.
Protective agent
In the present invention, protective agent refers to the reagent for protecting marker activity to improve labeled particle stability.
For protectant type, the present invention is not particularly limited, and can be made common any normal in diagnostic reagent field
The protective agent seen.Illustrative protective agent can be selected from carbohydrate protective agent, such as sucrose, trehalose, glucan, glucose, fructose；
Protide protective agent, such as BSA, casein, skimmed milk power；Alcohols protective agent, such as mannitol；For protectant dosage,
Those skilled in the art can select according to protectant type, for example, 5~100g/L can be used when using trehalose
Concentration.
Preservative
In the present invention, preservative refers to the reagent for extending the labeled particle holding time.
For the type of preservative, the present invention is not particularly limited, and can be used common any in diagnostic reagent field
Common preservative.Illustrative preservative can be selected from Sodium azide, phenol, P-hydroxybenzoic acid, benzoic acid, sodium benzoate, mountain
Pears acid, potassium sorbate, calcium propionate, dimethlbenzene, paraben esters, permanganate, antibiotics (such as gentamicin, dichloroacetyl
ProClin series etc. that amine, SUPELCO company release), one of ethyl-para-hydroxybenzoate and ethyl mercury sodium thiosulfate
Or it is several.
For the dosage of preservative, those skilled in the art can select according to the type of preservative, for example,
The concentration of such as 0.1~10g/L can be used when using ProClin300.
Surfactant
In the present invention, surfactant refers to that the interface state that can make its solution system on a small quantity, which is added, occurs significant change
Substance；It has fixed hydrophilic lipophilic group and can align on the surface of solution.
For kinds of surfactants, the present invention is not particularly limited, and can be used in diagnostic reagent field common
What common surfactant.Illustrative surfactant can be selected from Tween, SPAN, Qula is led to, EMULGEN series surface
Activating agent (such as polysorbas20, polysorbate40 and Triton X-100) and the more glycosides of Gelucire 44/14, anionic alkyl group, ten
One or more of dialkyl dimethyl glycine betaine, 3ARAMT1, SHO62C and AM PHITOL.
For the dosage of surfactant, those skilled in the art can select according to the type of surfactant
It selects, for example, the concentration of such as 1~20g/L can be used when using Triton X-100.
Labeling method
The present invention provides a kind of method for marking microballoon, the described method comprises the following steps：
1) microballoon and object to be marked are diluted using buffer；
2) mixed solution of the microballoon Yu object to be marked is obtained；
3) crosslinking agent is added to be incubated for；
4) blocking agent is added；
5) labeled microballoon is obtained；
Wherein, the microballoon is carboxyl microballoon；The pH of buffer in step 1) is 6.3~9.5.
In the method for the invention, step 1) can carry out in the following manner：
It is molten to obtain microspheres solution and object to be marked that microballoon and object to be marked are diluted respectively using buffer solution A and buffer solution B
Liquid, wherein buffer solution A and buffer solution B can be identical or be different；Or
Object to be marked is first added directly into the microspheres solution later to obtain microspheres solution using buffer dilution microballoon
In.
When diluting microballoon and object to be marked respectively, the concentration of microspheres solution may be, for example, 0.01~100mg/ml, may be used also
For 0.1~50mg/ml；And the concentration of object solution to be marked may be, for example, 0.1~100mg/ml, can also be 0.1~30mg/ml.
In some embodiments, object to be marked can come relative to 0.1%~30% weight ratio of microballoon using this is heavy
Amount can also be 0.5%~25%, can also be 1%~15%.
As described in the method for the present invention, in this method before the step 4) do not include it is any activated using activator it is micro-
The step of ball.
The crosslinking agent of any carboxyl microballoon may be incorporated in method of the invention in this field.Illustrative crosslinking agent packet
It includes but is not limited to EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), DCC (dicyclohexylcarbodiimide)
With DIC (N, N'- diisopropylcarbodiimide), they are dry powder form in general.In some preferred embodiments,
Crosslinking agent is preferably come with 0.5~4% weight ratio relative to microballoon using demonstrated in embodiment as follows, in the present invention
Method when using the crosslinking agent usage amount, will further facilitate to obtain the carboxyl microballoon that label works well.In addition, crosslinking
Agent more preferably comes using, the weight ratio with 1.0%~3.5% weight ratio relative to microballoon again or such as 1.0%, 1.5%,
2%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%,
3.7%, 3.8%, 3.9% or 4.0%.
Indicated by as the following examples, in the method for the invention without carrying out any purifying or dispersion steps.Its
In, purification step for example may include：The purification step that free activator is removed after activating to microballoon is walked in label
The purification step of free object to be marked is removed after rapid；And dispersion steps may include：To after centrifugation microballoon or agglutination after
Microballoon the step of being resuspended.Common purification step is for example, by using modes such as centrifugation (low temperature) or ultrafiltration, and common dispersion
For step for example, by using the modes such as ultrasound or oscillation, these above-mentioned modes are by the inevitable extension reaction time and improve to anti-
Answer environment and equipment requirement.And method of the invention then overcomes drawbacks described above.
The incubation process of step 3) is actually (even by the label of the activation of carboxyl microballoon and microballoon in method of the invention
Connection) synchronize progress.During incubation, incubation time can be more than or equal to 10 minutes, for example, 10 minutes, 20 minutes, 1 hour, 2
Hour, 4 hours etc.；The temperature of incubation can be 2~80 DEG C, for example, room temperature (25 DEG C).
In the step 4) of method of the invention, closing can be carried out for example at least 10 minutes, for example, 10 minutes, 20 points
Clock, half an hour, 1 hour etc..
In some embodiments, method of the invention is between the step of closing step and obtaining labeled particle
The step of one of protective agent, preservative and surfactant or more is added.When be added it is a variety of when can using simultaneously,
The mode of order or combinations thereof carries out.
Kit
It can be used for preparing immunity detection reagent through microballoon labeled caused by the method for the present invention, wherein immune inspection
Survey may be, for example, latex Immunoturbidimetric kit, fluorescence immunoassay detection kit, chemiluminescence detection kit etc..
For example, in one embodiment, labeled microballoon caused by method of the invention can be used as immunoturbidimetry
Detection reagent (R2) in kit, in this case, which can also include for samples of incubation
Reagent (R1) can also include for making calibration object/caliberator of working curve, can also include the matter for Quality Control control
Control product/Quality Control object etc..
In yet another embodiment, the labeled microballoon that method of the invention generates can be micro- for the carboxyl with fluorescence
Ball, after antigen or antibody mark fluorescent microballoon by sample antibody or antigen capture formed the antigen-antibody with fluorescence it is compound
Object, then the strong and weak concentration that can calculate determinand in sample for passing through detection fluorescence signal.
In yet another embodiment, the labeled microballoon that method of the invention generates can be micro- for magnetic carboxyl
Ball (i.e. magnetic bead) resists accordingly when that can combine in sample after the antigen for the substance markers that shine or antibody are tagged on magnetic bead
Body or antigen form magnetic bead antigen antibody complex, add substrate and cause chemiluminescence, by detecting chemiluminescence signal value
The concentration of determinand can be calculated.
Immune reagent kit of the invention can be used for various determinands, and illustrative kit includes：Retinol combination egg
White detection kit, β2-microglobulin detection kit, cystatin C detection kit, CRP detection kit, serum amyloid sample
Protein detection kit, lipoprotein detection kit, antistreptolysin O (ASO) detection kit, myoglobin assay kit
Deng.However, the present invention is not limited thereto.
Analysis method
Instrument and material
It is formulated for the R1 reagent of samples of incubation：
PH is adjusted to 7.4 ± 0.1.
R2 reagent of the preparation for detection：Using labeling method of the invention, specifically see following Examples 1 and 2.
Instrument：7180 automatic clinical chemistry analyzer of Hitachi.
Instrument parameter setting：
Method：2 end-point methods；
1/ reagent 2 of sample/reagent is respectively 2 μ l/155 μ l/25 μ l；
Dominant wavelength：546nm；
Commplementary wave length：700nm；
Reaction time：10min；
Survey luminous point：19~34 points；
Reaction temperature：37℃.
Calibration object (cystatin C)：Standard items 1,0.00mg/L；Standard items 2,0.50mg/L；Standard items 3,1.00mg/L；
Standard items 4,2.00mg/L；Standard items 5,4.00mg/L；Standard items 6,8.00mg/L.
Quality-control product (cystatin C)：Quality-control product level 1, sign value 0.86 (0.68~1.04) mg/L；Quality-control product level 2, mark
Indicating value 3.8 (3.04~4.56) mg/L.
Cystatin C high level sample：Human serum sample, cystatin C concentration about 36mg/L
Calibration curve signal measuring
Calibration is carried out to the reagent R2 made using automatic clinical chemistry analyzer and the measurement of quality-control product is analyzed, it is specific to tie
Fruit is see following Examples 1 and 2.
Sensitivity for analysis measurement
Using calibration object 6 and 5 replication of calibration object 2 times, the average value of absorbance difference is calculated, 1.00mg/L is scaled
Δ A, calculation formula is as follows：
In formula：
Indicate the average value of Cal-6 absorbance difference；
Indicate the average value of Cal-5 absorbance difference；
The mark concentration of C6 expression Cal-6；
The mark concentration of C5 expression Cal-5.
Judged according to scaling results, if the Δ A of 1.00mg/L is higher, indicates that sensitivity for analysis is higher, reagent is high in measurement
There is better reaction gradient when holding concentration calibration product, while also showing that the label effect of reagent is better indirectly, as a result see under
State Examples 1 and 2.
CV measurement in batch
Distinguished replication 10 times using Quality Control object level 1 and Quality Control object level 2, its variation within batch is calculated as follows
Coefficient (CV)：
In formula：
CV indicates the coefficient of variation；
S indicates standard deviation；
Indicate measurement mean value.
Reagent repeatability difference is judged according to CV value, and the smaller expression reagent repeatability of CV is better, on the contrary then poorer.
Equivalence zone measurement
Cystatin C high level sample is diluted with physiological saline, is prepared a series of containing various concentration antigen (reason
By concentration be 4.00mg/L, 8.00mg/L, 16.00mg/L, 20.00mg/L, 25.00mg/L, 30.00mg/L, 32.00mg/L,
34.00mg/L, 36.00mg/L) sample；It is successively measured from low concentration to high concentration later, the trend of measurement result is
First increase reduces afterwards, when measure concentration be lower than the range of linearity upper limit (8.00mg/L) for the second time when can terminate experiment, then with
Sample theory concentration is abscissa, and measured concentration draws dose-effect curve figure, when measured concentration occurs again as ordinate
When suitable with range of linearity upper concentration (8.00mg/L), which is the equivalence zone of the reagent.Experimental result
See following embodiments 3, table 14 and Fig. 3.
Long-time stability measurement at 2~8 DEG C
Visual inspection：About 3ml is taken out in test tube after prepared reagent is mixed, and Seal and preservation is placed onIt is taken out at point 0 day, 6 months, 12 months and reagent is visually observed to light, if milky suspension, without precipitating then table
Show that reagent appearance is normal, reagent stability is further determined by quality-control product measurement result；Otherwise it is indicated if reagent has precipitating
Reagent stability is not good enough, further determines whether reagent stability is subjected to by quality-control product measurement.
Quality-control product measuring method：After using series of calibration product calibrating reagent, point 0 day, 6 months, measurement Quality Control in 12 months
Average value is calculated separately after product level 1 and level 2 (every group of three repetitions), is then calculated by the following formula to obtain relative deviation
(%)：
(the measurement average value for saving Quality Control level when measuring average value -0 day of Quality Control level after a certain period of time)/0
It when Quality Control level measurement average value × 100%.
When relative deviation and First Determination result difference exceed ± 10%, it is generally recognized that reagent stability is not good enough and can not
Receive.
The present invention is described in more detail by the following examples, but the present invention is not limited to these Examples.
Embodiment 1 uses Good ' s buffer to prepare labeled carboxyl microballoon
Conventional method A
A kind of current usually used carboxyl microballoon labeling method is as follows：
1) the anti-human cystatin C polyclonal antibody of acetate dissolution (the Sichuan mikey biological material skill of 50mM pH 6.0 is used
Art Co., Ltd) point to antibody concentration be 1mg/mL；
2) the MES suspended microspheres for using 50mM pH 6.0, make its concentration 1%w/v；
3) antibody-solutions of monoploid product are added to carboxyl microballoon (the 98nm latex, Sichuan of 10 times of volumes while stirring
Mikey biology new material technology Co., Ltd) in suspension, persistently stir 20 minutes at room temperature；
4) EDC solution is added, so that EDC and microspheres weight ratio are 5%；
5) pH to 7.4 at room temperature, is adjusted immediately, mixes reaction 30 minutes；
6) centrifugation removal supernatant, with store buffer liquid (2g/L Tris, 10g/L sucrose and 5g/L sorbic acid) ultrasound weight
It is outstanding.
As shown in the above, this method needs the step of pH value is adjusted after addition of the cross-linking agent；Simultaneously in order to anti-
Only microballoon is aggregated, and this method needs to be centrifuged after cross-linking to remove the activator to dissociate in supernatant and the unbonded antibody that dissociates,
It also needs to carry out microballoon ultrasound and step is resuspended so that it disperses.Accordingly, there exist operating process complicated and time consumption, need large scale equipment
The problems such as being unfavorable for industrialization.
Accordingly, the present invention has carried out following experiment.
Experiment 1
Method：
Carboxyl microballoon and anti-human cystatin C polyclonal antibody are not diluted to using the HEPES buffer solution of 30mM, pH6.3
20mg/ml and 30mg/ml；Antibody-solutions after dilution and the microspheres solution after dilution are mixed, so that the weight of antibody and microballoon
Amount is than being 14~16%.
EDC mother liquor is added into mixed solution, so that the weight ratio of EDC and microballoon is 5%, and in 25 DEG C of subscripts
Note 30 minutes.
TRIS (final concentration 2g/L) is then added to close 30 minutes.
Sucrose (final concentration 10g/L) and sorbic acid (5g/L) is added, obtains the microballoon for being marked with antibody after standing 30 minutes.
Experiment 2~6 and Comparative examples A 1
In comparison with experiment 1, the operating parameter of experiment 2~6 and Comparative examples A 1 is given in the following table 1, wherein the ginseng not provided
Number is identical as experiment 1.
Table 1
As a result：
For confirmatory experiment 1~6 and the dispersibility of reagent prepared by Comparative examples A 1 and conventional method A mentioned above, react
The effect of property and labeling effciency etc., observes the appearance in its labeling process, as a result as shown in Figure 1.By the figure
As it can be seen that the experimental group for testing 4 keeps clarification in labeling process, and conventional method A and Comparative examples A 1 are in labeling process
There is serious agglutination.Further, according to the description in above-mentioned " analysis method " part, to calibration curve signal and analysis
Sensitivity is determined.As a result as shown in table 2 below.
Table 2
Note：It is the blank absorbency of reagent reaction end in bracket
As shown in Table 2, slow using the Good ' s of pH6.3~9.5 firstly, when being marked using method of the invention
The experiment 1~6 of fliud flushing keeps clear state in labeling process；There is agglutination phenomenon during the preparation process in conventional method A, subsequent
Centrifugation has been carried out in experimentation and operation is resuspended in ultrasound；And the Comparative examples A 1 that pH of buffer is 6.0 is same in labeling process
There is agglutination serious situation, it must not be without centrifugation and ultrasonic resuspension processing during subsequent experimental.Secondly, by calibrating
The blank absorbency that curve signal result can be seen that the reaction end of experiment 1~6 is far smaller than conventional method A and comparative example
A1 illustrates that the dispersibility of the obtained reagent of experiment 1~6 is more preferable；In addition, in each school in 0.50,1.00,2.00,4.00 and 8.00
Locate on schedule, for the absorbance of experiment 1~6 obviously higher than conventional method A and Comparative examples A 1, this proves the reaction of experiment 1~6 more
Strongly, label effect is more excellent.Finally, by sensitivity for analysis result it is found that the sensitivity for analysis of experiment 1~6 is apparently higher than routine
Method A and Comparative examples A 1, that is to say, that reaction reagent obtained by experiment 1~6 has more preferably labeling effciency.
Further, by comparing known to the calibration curve and sensitivity for analysis experimental result of experiment 1~6：Relative to pH6.3
For the buffer of pH9.5, the reagent that pH is prepared when being 7.0~9.0 has more preferably dispersibility, labeling effciency；And in pH
When being 7.8~8.5, the dispersibility of reagent reaches best with labeling effciency.
In addition, can also be seen that by comparing the result of experiment 1~6：It is 3mM's or 30mM relative to ionic strength
For Good ' s buffer, the reagent of the ionic strength preparation of 5mM~25mM has more preferably dispersibility, labeling effciency；And
Under the ionic strength of 10mM~20mM, the dispersibility of reagent reaches best with labeling effciency.
Comparative examples A 2
Method：
In order to confirm that method of the invention can achieve more preferably effect when using 3~30mM Good ' s buffer, into
Comparative examples A 2 is gone, the ionic strength of buffer used by the comparative example is 50mM, remaining experiment condition is identical as testing 3.
As a result：
For the dispersibility of reagent prepared by comparison example A2, reactivity and label effect etc., in its labeling process
Appearance observed, as seen from Figure 1, there is serious agglutination in the microballoon in labeling process of Comparative examples A 2；Simultaneously according to above-mentioned
Description in " analysis method " part, is determined calibration curve signal and sensitivity for analysis.As a result such as the following table 3 institute
Show.
Table 3
Note：It is the blank absorbency of reagent reaction end in bracket
As shown in Table 3, when being marked using method of the invention, using the Good ' s of 3mM~30mM ionic strength
Good ' s buffer of the buffer compared to 50mM can reach more preferably dispersibility, reactivity and label effect.
Experiment 7~10
Method：
The experiment 3 in experiment 1~6 to be chosen, carries out experiment 7~10 in the method, the parameter of change is as shown in table 4 below,
Remaining experiment condition is identical as experiment 3.
Table 4
| The weight ratio of EDC and microballoon | |
| Experiment 3 | 5.0% |
| Experiment 7 | 4.0% |
| Experiment 8 | 3.0% |
| Experiment 9 | 1.0% |
| Experiment 10 | 0.5% |
As a result：
For the dispersibility of reagent prepared by confirmatory experiment 7~10, reactivity and label effect etc., according to above-mentioned " analysis
Description in method " part is determined calibration curve signal and sensitivity for analysis.As a result as shown in table 5 below.
Table 5
Note：It is the blank absorbency of reagent reaction end in bracket
As shown in Table 5, by test 3 method and its parameter on the basis of by the weight ratio of EDC and microballoon by 5.0%
It is adjusted to 0.5%~4.0%, the dispersibility, reactivity and label effect of prepared reagent can be advanced optimized.Wherein, when
The weight ratio of EDC and microballoon reaches optimal effect when being 3.0% or so.
Experiment 11~13
Method：
It chooses effect in experiment 7~10 and preferably tests 8, continue experiment 11~13, the ginseng of change in the method
Number is as shown in table 6 below, remaining experiment condition is identical as experiment 8.
Table 6
| Buffer type | Microspherulite diameter nm | |
| Experiment 8 | MOPS | 98 |
| Experiment 11 | MOPS | 300 |
| Experiment 12 | TAPS | 98 |
| Experiment 13 | HEPPSO | 98 |
As a result：
For the dispersibility of reagent prepared by confirmatory experiment 11~13, reactivity and label effect etc., according to above-mentioned " analysis
Description in method " part is determined calibration curve signal and sensitivity for analysis.As a result as shown in table 7 below.
Table 7
Note：It is the blank absorbency of reagent reaction end in bracket
As shown in Table 7, right in the partial size for having adjusted microballoon, or after buffer is changed to other Good ' s buffers
Dispersibility, reactivity and the label effect of reagent prepared by method of the invention do not influence significantly.
Embodiment 2 prepares labeled carboxyl microballoon using amino acids buffer
Conventional method B
A kind of current usually used carboxyl microballoon labeling method is as follows：
1) anti-human cystatin C polyclonal antibody (Sichuan Michelson is dissolved using the glycine buffer of 500mM pH 6.0
Object new material technology Co., Ltd) to antibody concentration be 1mg/mL；
2) glycine buffer suspended microspheres (126nm latex, the Sichuan mikey biological material skill of 500mM pH 6.0 are used
Art Co., Ltd), make its concentration 1%w/v；
3) antibody-solutions of monoploid product are added in the microsphere suspensions of 10 times of volumes while stirring, are persistently stirred at room temperature
It mixes 20 minutes；
4) EDC solution is added, so that EDC and microspheres weight ratio are 5%；
5) pH to 7.4 at room temperature, is adjusted immediately, mixes reaction 30 minutes；
6) centrifugation removal supernatant, with store buffer liquid (2g/L Tris, 10g/L sucrose and 5g/L sorbic acid) ultrasound weight
It is outstanding.
Experiment 21
Method：
Using the glycine buffer of 500mM, pH6.3 by carboxyl microballoon (126nm latex, Sichuan mikey biological material
Technology Co., Ltd.) with anti-human cystatin C polyclonal antibody be not diluted to 15mg/ml and 20mg/ml；By the antibody after dilution
Microspheres solution mixing after solution and dilution, so that the weight ratio of antibody and microballoon is 14~16%.
EDC mother liquor is added into mixed solution, so that the weight ratio of EDC and microballoon is 5%, and in 25 DEG C of subscripts
Note 20 minutes.
Casein (final concentration 5g/L) is then added to close 20 minutes.
Trehalose (final concentration 5g/L) and phenol (1g/L) is added, stands the microballoon for obtaining being marked with antibody after twenty minutes.
Test 22~26 and comparative example B
In comparison with experiment 21, the operating parameter of experiment 22~26 and comparative example B is given in the following table 8, wherein do not provide
Parameter is identical as experiment 21.
Table 8
| PH of buffer | Buffer type | Ionic strength mM | |
| Experiment 21 | 6.3 | Glycine | 500 |
| Experiment 22 | 7.0 | Arginine | 15 |
| Experiment 23 | 7.8 | Glycine | 40 |
| Experiment 24 | 8.5 | Arginine | 250 |
| Experiment 25 | 9.0 | Glycine | 300 |
| Experiment 26 | 9.5 | Arginine | 10 |
| Comparative example B1 | 6.0 | Glycine | 250 |
As a result：
For dispersibility, reactivity and the labeling effciency etc. of reagent prepared by confirmatory experiment 21~26 and comparative example B
Effect, the appearance in its labeling process is observed, as a result as shown in Figure 2.It may be seen that by taking experiment 24 as an example
Experimental group keeps clarification in labeling process, and serious agglutination occurs in labeling process in comparative example B.Simultaneously according to it is above-mentioned " point
Description in analysis method " part, is determined calibration curve signal and sensitivity for analysis.As a result as shown in table 9 below.
Table 9
Note：It is the blank absorbency of reagent reaction end in bracket
As shown in Table 9, firstly, when being marked using method of the invention, using the amino acids of pH6.3~9.5
The experiment 21~26 of buffer keeps clear state in labeling process；There is agglutination phenomenon during the preparation process in conventional method B, and
Centrifugation has been carried out during subsequent experimental and operation is resuspended in ultrasound；The comparative example B that pH of buffer is 6.0 is in labeling process
It is same agglutination serious situation occur, centrifugation and ultrasonic resuspension processing have also been carried out during subsequent experimental.Secondly, by calibrating
The blank absorbency that curve signal result can be seen that the reaction end of experiment 21~26 is far smaller than conventional method B and compares
Example B illustrates that the dispersibility of the obtained reagent of experiment 21~26 is more preferable；And in each school in 0.50,1.00,2.00,4.00 and 8.00
Locate on schedule, for the absorbance of experiment 21~26 obviously higher than conventional method B and comparative example B, this proves the reaction of experiment 21~26 more
For strong, label effect is more excellent.Finally, by sensitivity for analysis result it is found that the sensitivity for analysis of experiment 21~26 is apparently higher than
Conventional method B and comparative example B, this illustrates that reaction reagent obtained by experiment 21~26 has more preferably labeling effciency.
Further, by comparing known to the calibration curve and sensitivity for analysis experimental result of experiment 21~26：Relative to
For the buffer of pH6.3 and pH9.5, the reagent that pH is prepared when being 7.0~9.0 has more preferably dispersibility, labeling effciency；And
When pH is 7.8~8.5, the dispersibility of reagent reaches best with labeling effciency.
In addition, can also be seen that by comparing the result of experiment 21~26：It is 10mM or 500mM relative to ionic strength
Amino acids buffer for, the reagent of the ionic strength of 15mM~300mM preparation has more preferably dispersibility, label effect
Rate；And under the ionic strength of 40mM~250mM, the dispersibility of reagent reaches best with labeling effciency.
Experiment 27~30
Method：
The experiment 24 in experiment 21~26 is chosen, carries out experiment 27~30, the parameter of change such as the following table 10 in the method
Shown, remaining experiment condition is identical as experiment 24.
Table 10
| The weight ratio of EDC and microballoon | |
| Experiment 24 | 5.0% |
| Experiment 27 | 4.0% |
| Experiment 28 | 3.0% |
| Experiment 29 | 1.0% |
| Experiment 30 | 0.5% |
As a result：
For the dispersibility of reagent prepared by confirmatory experiment 27~30, reactivity and label effect etc., according to above-mentioned " analysis
Description in method " part is determined calibration curve signal and sensitivity for analysis.As a result as shown in table 11 below.
Table 11
Note：It is the blank absorbency of reagent reaction end in bracket
As shown in Table 11, by test 24 method and its parameter on the basis of by the weight ratio of EDC and microballoon by
5.0% is adjusted to 0.5%~4.0%, can advanced optimize the dispersibility, reactivity and label effect of prepared reagent.Its
In, reach optimal effect when the weight ratio of EDC and microballoon is 3.0% or so.
Experiment 31~33
Method：
The experiment 28 in experiment 27~30 is chosen, continues experiment 31~33 in the method, the parameter of change is as follows
Shown in table 12, remaining experiment condition is identical as experiment 28.
Table 12
| Buffer type | Microspherulite diameter nm | |
| Experiment 28 | Arginine | 126 |
| Experiment 31 | Arginine | 350 |
| Experiment 32 | Alanine | 126 |
| Experiment 33 | Lysine | 126 |
As a result：
For the dispersibility of reagent prepared by confirmatory experiment 31~33, reactivity and label effect etc., according to above-mentioned " analysis
Description in method " part is determined calibration curve signal and sensitivity for analysis.As a result as shown in table 13 below.
Table 13
Note：It is the blank absorbency of reagent reaction end in bracket
As shown in Table 13, in the partial size for having adjusted microballoon, or after buffer is changed to other amino acids buffers,
It still can reach the effect that the method for the present invention is realized.
3 performance test of embodiment
According to the description in " analysis method " part above, conventional method, experiment of the invention and comparative example criticize interior
Long-time stability at CV test, equivalence zone and 2~8 DEG C, to verify the effect of method of the invention when being used as diagnostic reagent.Knot
Fruit is as shown in table 14, Fig. 3.
Table 14
Note：" milky white " expression milky suspension, without precipitating
Compared to conventional method and comparative example it can be seen from table 14, Fig. 3, the tool of reagent prepared by experimental method of the invention
There is CV in lower crowd, so that repeatability is more preferable；Reagent prepared by experimental method of the invention simultaneously has broader equivalence zone,
Prove that method of the invention further improves the labeling effciency of microballoon and as the successive optimization labeling effciency of parameter gradually mentions
There is the probability of false positive or false negative in detection application so as to reduce in height；In addition, stability result is seen, it is of the invention
The absolute value of Quality Control relative deviation at 6 months and 12 months of reagent prepared by method is respectively less than 10%, has good stability；
And this field when marking microballoon used conventional method at 6 months when occurred precipitating on a small quantity, it is a large amount of heavy then to occur at 12 months
It forms sediment, stability is poor.
Claims (22)
1. a kind of method for marking microballoon, the described method comprises the following steps：
1) microballoon and object to be marked are diluted using buffer；
2) mixed solution of the microballoon Yu object to be marked is obtained；
3) crosslinking agent is added to be incubated for；
4) blocking agent is added；
Wherein, the microballoon is carboxyl microballoon；The pH of buffer in step 1) is 6.3~9.5.
2. according to the method described in claim 1, wherein, the pH of the buffer in step 1) is 7.0~9.0.
3. according to the method described in claim 1, wherein, the buffer in step 1) is the Good ' that ionic strength is 3~30mM
The amino acids buffer that s buffer or ionic strength are 10~500mM.
4. according to the method described in claim 3, wherein, Good ' the s buffer be selected from HEPES, MOPS, MES, TES,
One of TAPS and HEPPSO or more.
5. according to the method described in claim 3, the ionic strength of Good ' the s buffer is 5mmol/L~25mmol/L.
6. according to the method described in claim 3, wherein, the amino acids buffer is neutral fat race buffered with amino acid liquid
And/or basic amine group acid buffer.
7. according to the method described in claim 6, wherein, the neutral fat race buffered with amino acid liquid is glycine buffer
And/or alanine buffer.
8. according to the method described in claim 6, wherein, the basic amine group acid buffer is Arginine buffer and/or relies
Propylhomoserin buffer.
9. according to the method described in claim 3, wherein, the ionic strength of the amino acids buffer be 15mmol/L~
300mmol/L。
10. method according to claim 1 to 9, wherein the weight ratio of the crosslinking agent and microballoon is
0.5%~4.0%, preferably 1.0%~3.5%.
11. method according to claims 1 to 9, wherein the object to be marked is amino-containing object to be marked.
12. according to the method for claim 11, wherein the amino-containing object to be marked is selected from protein, peptide and more
One of peptide or more.
13. according to the method for claim 11, the amino-containing object to be marked is antigen and/or antibody.
14. according to the method for claim 13, the antibody is monoclonal antibody and/or polyclonal antibody.
15. method according to claims 1 to 9, the method also includes：
5) one of protective agent, preservative and surfactant or more is added.
16. method according to claims 1 to 9, wherein the crosslinking agent is EDC, DCC or DIC, preferably EDC.
17. method according to claims 1 to 9, wherein the method does not include purification step.
18. according to the method for claim 17, wherein the purification step is carried out using ultrafiltration and/or centrifugation.
19. method according to claims 1 to 9, wherein the method does not include the steps that dispersion microsphere.
20. according to the method for claim 19, wherein carried out using ultrasound or vibrate the step of the dispersion microsphere.
21. the labeled microspheres solution being prepared by the method for any one of claim 1~20.
22. a kind of kit, the kit includes microspheres solution labeled described in claim 21.
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| CN113419059A (en) * | 2021-06-30 | 2021-09-21 | 迈克生物股份有限公司 | Blocking agent for chemiluminescence immune analysis method |
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