CN105403693B - A kind of preparation method of magnetic microparticle chemiluminescence reagent - Google Patents
A kind of preparation method of magnetic microparticle chemiluminescence reagent Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
Abstract
This disclosure relates to a kind of preparation method of magnetic microparticle chemiluminescence reagent.Further relate to a kind of detection kit of human muscle hemoglobin.In the method, magnetic particle is first mixed with crosslinking agent, later again with treating that coated antibody is combined.In addition, disclosed method additionally uses polymer closing magnetic particle.The detection reagent prepared by this method combines the double dominant of chemiluminescence and magnetic particle technology, and magnetic particle makes the kit have higher specificity, sensitivity, the detection range of bigger and quick detection time.
Description
Technical field
The disclosure belongs to immunological technique detection field more particularly to a kind of preparation side of magnetic microparticle chemiluminescence kit
Method and the kit prepared using the method；The disclosure further relates to a kind of quantitative reagent for detecting myoglobins in human serum
Box.
Background technology
Myoglobins be it is a kind of be present in cardiac muscle and Skeletal Muscle Cell matter protein, molecular weight 17.8kD.
After myocyte is damaged, myoglobins is released quickly against in the circulatory system.The myoglobins measured in serum is to examine
Disconnected acute myocardial infarction (AMI), block again and thromboembolism treatment after success pour into one of important indicator of diagnosis again.Concentration is in disease
About two hour myoglobin concentration raisings after shape occurs, therefore can be as the early stage index of diagnosing cardiac infarction.According to difference
Pour into remedy measures again, after infraction is formed and started, myoglobin concentration in blood circulation is 4 to 12 after infraction occurs
Hour can reach peak, drop to normal level after about 24 hours.After skeletal muscle is damaged and renal function serious hindrance also may be used
Myoglobin concentration is caused to increase.
In the case of no Skeletal muscle injury, myoglobins is used as the early sign of myocardial infarction illness.Myoglobins
Negative prediction the result is that 99%, for excluding non-Patients With Myocardial Infarction.When with reference to other cardiac markers (such as CK-MB or flesh
Calcium protein I) together in application, myoglobins has remarkable diagnostic value, available for assessing potential acute myocardial infarction trouble
Person.
The myoglobins detection means clinically used at present includes：ELISA method and immunoturbidimetry.However, these sides
The shortcomings that method, is that sensitivity is not high enough, and detection duration, the range of linearity is not wide enough, and sample has to be diluted processing.
In view of above-mentioned shortcoming, is developed a kind of new detection method, combines magnetic separation technique and chemistry
A kind of myoglobins quantitative determination reagent kit is disclosed in the double dominant of luminescence technology, such as Chinese patent CN102435752B
And its detection method, the kit include and are marked with the magnetic microsphere of anti-myoglobins monoclonal antibody and alkaline phosphatase mark
The anti-myoglobins monoclonal antibody of note.Change method to be combined chemiluminescence with immune magnetic particle, so as to provide one
Kind has higher detection sensitivity and specificity close to homogeneous reaction system.However, antibody coating magnetic is micro- in the prior art
The method of grain is that magnetic particle and antibody are first added in crosslinking agent (such as EDC) after mixing.Even if in CN102435752B,
It is to be mixed antibody and crosslinking agent disuccinimidyl suberate in advance.In these method for coating, crosslinking agent can damage
The activity of evil antibody.Also, the reagent range of linearity is not broad enough, and high level sample needs are diluted processing in advance.In consideration of it, this
The technical staff in field is highly desirable to obtain a kind of novel magnetic particle method for coating to improve the defects of above-mentioned technology.
Invention content
According to the one side of the application, a kind of method for coating of magnetic particle is provided, including step：
A) magnetic particle and combination buffer are contacted, obtains the first mixture；
B) carbodiimide and the first mixture are contacted into 10min to 1h at 20 DEG C to 30 DEG C, obtains the second mixture；And
C) monoclonal antibody and the second mixture are protected from light 2h to 4h at 20 DEG C to 30 DEG C, obtain being coated with monoclonal
The magnetic particle of antibody.
According to some embodiments, the pH of the combination buffer is 4.5 to 6.0；Preferably, pH is selected from：4.5、4.8、
5.0th, the range between 5.2,5.5 and above-mentioned any two numerical value.In preferred embodiment, the combination buffering
The pH of liquid is 5.0.In a particular embodiment, the combination buffer is MES buffer solutions.In preferred embodiment
In, combination buffer is the 0.1M MES buffer solutions that pH is 5.0.
According to some embodiments, the grain size of the magnetic particle is 1 μm to 3 μm.In a preferred embodiment, the magnetic
The grain size of particle is 0.8 to 1.2 μm.Technical staff understands that it is practically impossible to be single for magnetic particle used in method of disclosure
A magnetic particle, but numerous magnetic particles.Therefore, the grain size of magnetic particle is the distribution of a particle size range statistically.
For example, when referring to the magnetic particle that grain size is 1 μm, the grain size for being not meant to each single magnetic particle is just 1 μm, and
It is to allow within the scope of 1 μm nearby (such as ± 10% error).In consideration of it, in the context of this application, with grain size
Range represent the grain size of magnetic particle.
According to some embodiments, by carbodiimide and the first mixture in 20 DEG C to 30 DEG C contacts in step b)
20min to 40min obtains the second mixture.In a preferred embodiment, by carbodiimide and the first mixture in step b)
In 22 DEG C to 28 DEG C mixing 25min to 35min.In a particular embodiment, a concentration of 8mg/ml to 15mg/ of carbodiimide
Ml, preferably 10mg/ml.
According to some embodiments, monoclonal antibody and the second mixture are protected from light at 22 DEG C to 28 DEG C in step c)
3h.In a particular embodiment, monoclonal antibody is myoglobins monoclonal antibody.However technical staff understands, although this
Specific monoclonal antibody is employed in the specific example of application, but the antibody of any other type is equally applicable to this Shen
Please.Because the present processes refer to a kind of improved magnetic particle coating step and closing step, as long as antibody can be tied
Magnetic particle is bonded to, is not only restricted to the specific structure (sequence of such as CDR) of antibody.
According to some embodiments, the method for coating of the magnetic particle of the application further includes step d)：List is coated with to described
Biolipidure is added in the magnetic particle of clonal antibody, 2 are carried out at 22 to 28 DEG C to 3h cappings；Later in 37 DEG C of reactions
15min to 60min.Optionally, the method for coating of the magnetic particle of the application further includes step e)：The closing terminated in step d) is anti-
It should.Optionally, the method for coating of the magnetic particle of the application further includes step f)：It centrifuges and collects the magnetic for being coated with monoclonal antibody
Particle.It is a kind of polymer with phosphatide polar group 2- methacryls phosphocholine (MPC)
(K.Ishihara et al. J.Biomed.Mater.Res., 39,323,1998 years).Including but not limited to
Biolipidure-103、Biolipidure-203、Biolipidure-206、Biolipidure-405、Biolipidure-
502nd, Biolipidure-702, Biolipidure-802, Biolipidure-1002, Biolipidure-1201 and
Biolipidure-1301.In the context of this application, Ren HeheEquivalent product can be equally used for
The present processes.In a particular embodiment, it is describedBe Biolipidure-502 or
Biolipidure-1002；It is preferred that Biolipidure-1002.
According to some preferred embodiments, in step d), add to described be coated in the magnetic particle of monoclonal antibody
Enter Biolipidure, 2 are carried out at 22 to 28 DEG C to 3h cappings, reacts 15min, 30min or 60min at 37 DEG C later, most
It is preferred that react 30min.
According to some specific embodiments, 0.5 is protected from light to 1.5h at 22 to 28 DEG C by adding in quenching buffers
To terminate capping.Quenching buffers well known in the art can use.For example, it in a particular embodiment, is quenched
Buffer solution includes 10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4.
According to a specific embodiment, the method for coating of magnetic particle includes step：
A) the 0.1M MES buffer solutions of 2 μm of magnetic particles and pH 5.0 are contacted, obtains the first mixture；
B) 10mg/ml carbodiimides and the first mixture are contacted into 30min at 20 DEG C to 30 DEG C, obtains the second mixture；
C) monoclonal antibody and the second mixture are protected from light 3h at 20 DEG C to 30 DEG C, obtain being coated with monoclonal antibody
Magnetic particle；
D) Biolipidure-1002 is added in described be coated in the magnetic particle of monoclonal antibody, in 22 to 28 DEG C of progress
3h cappings react 30min at 37 DEG C later；
E) quenching buffers are added in and at 20 DEG C to 30 DEG C are protected from light 1h to terminate the capping, it is described be quenched it is slow
Fliud flushing includes 10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4；
F) it centrifuges and collects the magnetic particle for being coated with monoclonal antibody.
According to the another aspect of the application, a kind of magnetic particle for being coated with monoclonal antibody is provided, is by this Shen
What method please was prepared.
According to the another aspect of the application, additionally provide and resisted by the monoclonal that is coated with that the present processes are prepared
Purposes of the magnetic particle of body in detection reagent is prepared.
According to the another aspect of the application, a kind of detection reagent is additionally provided, monoclonal is coated with it includes the application
The magnetic particle of antibody.
According to the another aspect of the application, a kind of myoglobin assay kit is additionally provided, it includes or by as follows
Composition：Magneto separate reagent R1, labelled reagent R2 and substrate solution.In a specific embodiment, Magneto separate reagent R1 includes coating
There is the magnetic particle of myoglobins monoclonal antibody.In R1, being coated with the magnetic particle of myoglobins monoclonal antibody allows to be placed in
In appropriate buffer solution.The buffer solution of R1 preferably comprises：0.6g/100ml NaCl, 0.2g/100ml polysorbas20s, 0.2g/100ml
NaN3,50mM Tris-HCl buffer solutions, pH7.6.A concentration of 2mg/ml of the myoglobins monoclonal antibody in R1.
In specific embodiment, labelled reagent R2 includes the myoglobins monoclonal antibody of enzyme label.In preferred embodiment,
Labelled reagent R2 includes the myoglobins monoclonal antibody and buffer solution of alkali phosphatase enzyme mark.Buffer solution preferably comprises：0.6g/
100ml NaCl, 0.2g/100ml polysorbas20s, 0.2g/100ml NaN3,50mM Tris-HCl buffer solutions, pH7.6.The alkali
A concentration of 0.1 μ g/ml of the myoglobins monoclonal antibody of acid phosphatase label in R2.
In a specific embodiment, substrate solution includes the substrate of enzyme, such as chemiluminescent substrate.
In other embodiments, the myoglobin assay kit of the application also includes myoglobins standard items.Mark
Quasi- product can be commercially available acquisitions or voluntarily prepare；Wherein include the myoglobins of known concentration.Technical staff is clear
Chu, the standard items purpose in kit are to draw a standard curve.Therefore, any calibration object that can cover detection range
Concentration can use.The concentration of myoglobins is selected from, but not limited to, following one or more：0、10、50、100、300、800、
The human muscle hemoglobin of 1500ng/ml.
In other embodiments, the myoglobin assay kit of the application also includes washing lotion.Preferably, washing lotion contains
There are polysorbas20, sodium chloride and PC300.
In a particular embodiment, it is coated on the magnetic particle of myoglobins monoclonal antibody and is closed with Including but not limited to Biolipidure-103, Biolipidure-203,
Biolipidure-206、Biolipidure-405、Biolipidure-502、Biolipidure-702、Biolipidure-
802nd, Biolipidure-1002, Biolipidure-1201 and Biolipidure-1301.In the context of this application, appoint
He HeEquivalent product can be equally used for the technical solution of the application.In a particular embodiment, institute
It statesIt is Biolipidure-502 or Biolipidure-1002；It is preferred that Biolipidure-1002.
In a particular embodiment, the magnetic particle for being coated with myoglobins monoclonal antibody is by the application
What method for coating was prepared.
Description of the drawings
Fig. 1：The comparison of the application method for coating and contrast method.
◆ representation method 1 be coated with after magnetic bead, each test the magnetic bead amount that uses be 20 μ g (y=533.72x+1.7E+5,
r2=0.8352)；
■ representation methods 1 be coated with after magnetic bead, each test the magnetic bead amount that uses be 40 μ g (y=1326.5x+1.8E+5,
r2=0.9656)；
▲ representation method 2 be coated with after magnetic bead, each test the magnetic bead amount that uses be 10 μ g (y=525.31x+36708,
r2=0.9734).
Fig. 2：Detection kit and the correlation of the kit of ripe listing compares prepared by the application method.It is related
Property equation be y=1.0858x-4.8577, r2=0.9836.
Specific embodiment
Embodiment
Embodiment 1：Contrast method
1. prepare the coated magnetic particle of myoglobins monoclonal antibody
1.1 abundant mixing magnetic particles (Merck, 0.8 to 1.2 μm of grain size, article No. 39433087), take 10mg to be placed in test tube
In, Magneto separate 1 minute removes supernatant.1ml combination buffers (0.1M MES, pH 5.0) are added in, Magneto separate 1 minute after mixing
After remove supernatant, add in mixing magnetic particle after 1ml combination buffers.
1.2 add in 100 μ g myoglobins monoclonal antibodies, room temperature (22 DEG C to 28 DEG C) mixing 30 minutes.
1.3 100 μ l 10mg/ml EDC of addition (face with now matching, solvent is the combination buffer of ice) room temperature and are protected from light mixing 3
Hour.Magneto separate removes supernatant, adds in dcq buffer liquid (50mM TBS, pH 7.4), and mixing Magneto separate removes supernatant, and repeatedly three
It is secondary.
1.4 addition 1ml quenching buffers (10mM PBS, pH 7.4, BSA containing 1g/100ml, 40mM glycine) room temperatures are kept away
Light mixing 1 hour, Magneto separate removal supernatant.
1.5 add in 1ml magnetic particles storing liquid (100mM PBS, pH 7.4, Tween 80 containing 0.1g/100ml, 1g/100ml
BSA), mixing Magneto separate removal supernatant.Repeatedly for three times, 4C in storing liquid is finally placed in save backup.
2.R1 preparation
With dilution (0.6g/100ml NaCl, 0.2g/100ml polysorbas20s, 0.2g/100ml NaN3,50mM Tris-
HCl buffer solutions, pH 7.6) magnetic particle 1 that will mark:5 dilutions are to get to R1 reagents.
Embodiment 2：Magnetic particle method for coating after optimization
1. prepare the coated magnetic particle of myoglobins monoclonal antibody
1.1 abundant mixing magnetic particles (Merck, 0.8 to 1.2 μm of grain size, article No. 39433087), take 10mg to be placed in test tube
In, Magneto separate 1 minute removes supernatant.1ml combination buffers (0.1M MES, pH 5.0) are added in, Magneto separate 1 minute after mixing
After remove supernatant, add in mixing magnetic particle after 1ml combination buffers.
1.2 100 μ l 10mg/ml EDC of addition (face with now matching, solvent is the combination buffer of ice), and room temperature is (at 22 DEG C extremely
28 DEG C) mixing 30 minutes.
1.3 add in 100 μ g myoglobins monoclonal antibodies, and room temperature is protected from light mixing 3 hours.Magneto separate removes supernatant, adds in rinse and delay
Fliud flushing (50mM TBS, pH 7.4), mixing Magneto separate removes supernatant, repeatedly for three times.
1.4 addition Biolipidure-1002 carry out closing 3h in room temperature, and are placed on 37 DEG C of processing 30min.
1.5 addition 1ml quenching buffers (10mM PBS, pH 7.4, BSA containing 1g/100ml, 40mM glycine) room temperatures are kept away
Light mixing 1 hour, Magneto separate removal supernatant.
1.6 add in 1ml magnetic particles storing liquid (100mM PBS, pH 7.4, Tween 80 containing 0.1g/100ml, 1g/100ml
BSA), mixing Magneto separate removal supernatant, repeatedly for three times, is finally placed in 4C in storing liquid and saves backup.
2.R1 preparation
With dilution (0.6g/100ml NaCl, 0.2g/100ml polysorbas20s, 0.2g/100ml NaN3,50mM Tris-
HCl buffer solutions, pH 7.6) magnetic particle 1 that will mark:5 dilutions are to get to R1 reagents.
3. result
The antibody coating magnetic particle method (contrast method) of embodiment 1, is first to be uniformly mixed magnetic particle and antibody, then
Antibody activity can be damaged by adding in crosslinking agent EDC, EDC.In order to protect antibody activity, the optimization method of embodiment 2 is employed.Experiment
As a result (Fig. 1) shows：Antibody activity is good in the method for embodiment 1, but linear high point ratio is limited, needs the magnetic after coating
Particle dosage is increased to 40 μ g from 20 μ g of each test can just obtain preferable high point ratio, this illustrates that coating efficiency is poor.It is real
The method general performance for applying example 2 more preferably, is each tested using the magnetic particle after 10 μ g coatings, is preferably linearly imitated with regard to that can reach
Fruit.
The closing of 3. magnetic particle of embodiment
In order to compare the sealing effect of confining liquid, after the step 1.3 of embodiment 2, the magnetic particle after coating is rushed
It is divided into two after wash buffer cleaning, is separately added into 200 μ l Biolipidure-502 or Biolipidure-1002 and is sealed
It closes.
As a result (table 1) shows that signal-to-noise ratio is 5.304 to the magnetic particle after Biolipidure-1002 closings in the reaction；It is sensitive
Degree is more preferable, and high level linear scale is also more preferable.Because Biolipidure is the polymer containing different molecular weight, make magnetic particle
Sealing effect is more preferable.
Magnetic particle after Biolipidure-1002 closings is placed in 37 DEG C and handles 15min, 30min and 60min respectively.Examination
It tests result (table 2) and shows the magnetic particle after handling 15min at 37 DEG C, performance in the reaction is with untreated without apparent
Difference.
And the magnetic particle after 30min is handled at 37 DEG C, best specificity and sensitivity are shown in the reaction, signal-to-noise ratio
Reach 7.224, the theoretical concentration of linear high level ratio and standard point closer to.
The magnetic particle after 60min is handled at 37 DEG C, background value has built up, and signal-to-noise ratio is caused to decline, and processing time mistake
Long, high level ratio is deteriorated.
The comparison of table 1.Biolipidure-502 and Biolipidure-1002 sealing effect
The comparison of table 2.Biolipidure-1002 difference off-period effects
Embodiment 4：The preparation of alkali phosphatase enzyme mark myoglobins antibody in R2 reagents
1mg alkaline phosphatases are taken, add in 0.5mg myoglobins monoclonal antibodies；Volume is mended to 500 with the 20mM PBS of pH 7.2
μ l, are put into bag filter, are placed in the 20mM PBS of pH 7.2 and dialyse three times.
10 μ l, 5% glutaraldehydes are added in after taking-up, room temperature is protected from light mixing 2 hours.It is again placed in the 20mM PBS of pH 7.2
Dialysis three times, removes glutaraldehyde, obtains the myoglobins monoclonal antibody of alkali phosphatase enzyme mark.
The antibody marked is stored in 50mM Tris-HCl (NaCl containing 0.9g/100ml, 0.1g/100ml NaN3)
In, the glycerine of equal volume is added in, packing is put into -20 DEG C and saves backup.
With dilution (0.6g/100ml NaCl, 0.2g/100ml polysorbas20s, 0.2g/100ml NaN3,50mM Tris-
HCl buffer solutions, pH 7.6) antibody 1 that will mark:5000 dilutions are to get to R2 reagents.
Embodiment 5：The preparation of human muscle hemoglobin standard items
The standard items concentrate purchased (being purchased from Fitzgerald) is diluted to working concentration with sheep serum, respectively
10、50、100、300、800、1500ng/ml。
Embodiment 6：It is prepared by washing lotion
Washing lotion is Tris-HCl buffer solutions, while also contains polysorbas20, a concentration of 0.6g/ of a concentration of 0.25g/100ml
The sodium chloride of 100ml and the PC300 of 0.1g/100ml.
Embodiment 7：Clinical test and performance indicator
Each reagent prepared by by embodiment 2 and 4 to 6 is assembled into the kit according to the disclosure.With the examination of the disclosure
Agent box carries out clinical test, and serum sample number is 458, from patient and Healthy People.
The myoglobins chemical luminescence reagent kit listed is had been approved by as contrast agents box using foreign countries；Its testing principle is
Double antibody sandwich method；Its kit forms is：Reagent 1 includes the magnetic particle of myoglobins labeling of monoclonal antibody, and reagent 2 includes
The myoglobins monoclonal antibody of acridinium ester label.
It is first detected with having been approved by the myoglobins chemical luminescence reagent kit of listing, then with this kit measurement.
As a result (Fig. 2) shows that the relationship equation of two kit measured values is y=1.0858x-4.8577, R2=0.9836, illustrate this
The measured value consistency of kit and commercialized product is preferable.
The kit of the disclosure realizes following performance indicator：
1) precision CV ＜ 10%；
2) deviations in accuracy ＜ 10%；
3) range of linearity is 10ng/ml to 1500ng/ml；
4) sensitivity is not less than 10ng/ml；
5) as bilirubin≤40mg/dl, hemoglobin≤1000mg/dl, during triglycerides≤1000mg/dl, measured value becomes
Change≤5%；
6) it is good that the reagent stability of 5 days is preserved at 37 DEG C, the standard items of 10 days are preserved at 37 DEG C and are had good stability；
7) difference between batch of the three batches of reagents successively prepared<10%.
The advantages of technical solution of the disclosure：The kit of the disclosure marks two monoclonal antibodies respectively in alkaline phosphorus
On sour enzyme and magnetic particle, wherein magnetic particle coated antibody ensure that better antibody activity and higher coating effect by optimization
Rate then closes magnetic particle using polymer and carries out heating treatment so that kit detects human serum in quasi- homogeneous reaction
Middle myoglobins has higher specificity, sensitivity, the detection range of bigger and quick reaction time.
Claims (16)
1. a kind of method for coating of magnetic particle, including step：
A) magnetic particle and combination buffer are contacted, obtains the first mixture；
B) carbodiimide and the first mixture are contacted into 10min to 1h at 20 DEG C to 30 DEG C, obtains the second mixture；
C) monoclonal antibody and the second mixture are protected from light 2h to 4h at 20 DEG C to 30 DEG C, obtain being coated with monoclonal antibody
Magnetic particle；
D) add in Biolipidure-1002 to described be coated in the magnetic particle of monoclonal antibody, 22 to 28 DEG C carry out 2 to
3h cappings react 30min at 37 DEG C later；
E) capping is terminated；
F) it centrifuges and collects the magnetic particle for being coated with monoclonal antibody；
Wherein, the monoclonal antibody is myoglobins monoclonal antibody.
2. according to the method described in claim 1, the pH of wherein described combination buffer is 4.5 to 6.0.
3. according to the method described in claim 2, the pH of wherein described combination buffer is selected from：4.5、4.8、5.0、5.2、5.5
And the range between above-mentioned any two numerical value.
4. according to the method described in claim 3, the pH of wherein described combination buffer is 5.0.
5. according to the method described in claim 1, wherein described combination buffer is MES buffer solutions.
6. according to the method described in claim 5, wherein described combination buffer is the 0.1M MES buffer solutions that pH is 5.0.
7. according to the method described in claim 1, the grain size of wherein described magnetic particle is 1 μm to 3 μm.
8. according to the method described in claim 1, the particle size range of wherein described magnetic particle is within 0.8 μm to 1.2 μm.
9. according to the method described in claim 1, wherein
Carbodiimide and the first mixture are contacted into 20min to 40min at 20 DEG C to 30 DEG C in step b).
10. according to the method described in claim 9, by carbodiimide and the first mixture in 22 DEG C to 28 DEG C mixings in step b)
25min to 35min.
11. the according to the method described in claim 1, wherein a concentration of 8mg/ml to 15mg/ml of step b) carbodiimides.
12. according to the method for claim 11, the wherein a concentration of 10mg/ml of step b) carbodiimides.
13. according to the method described in claim 1, by monoclonal antibody and the second mixture at 22 DEG C to 28 in wherein step c)
DEG C it is protected from light 3h.
14. according to the method described in claim 1, be protected from light by adding in quenching buffers at 22 DEG C to 28 DEG C 0.5 to
1.5h carries out the step e).
15. according to the method for claim 14, the quenching buffers include 10mM PBS, 1g/100ml BSA, 40mM
Glycine, and pH is 7.4.
16. according to the method described in claim 1, including step：
A) the 0.1M MES buffer solutions of 0.8 μm to 1.2 μm of magnetic particle and pH 5.0 are contacted, obtains the first mixture；
B) 10mg/ml carbodiimides and the first mixture are contacted into 30min at 20 DEG C to 30 DEG C, obtains the second mixture；
C) monoclonal antibody and the second mixture are protected from light 3h at 20 DEG C to 30 DEG C, obtain being coated with the magnetic of monoclonal antibody
Particle；
D) Biolipidure-1002 is added in described be coated in the magnetic particle of monoclonal antibody, 3h is carried out at 22 DEG C to 28 DEG C
Capping reacts 30min at 37 DEG C later；
E) quenching buffers are added in and to 30 DEG C are protected from light 1h at 20 DEG C to terminate the capping, the quenching buffers
Include 10mM PBS, 1g/100ml BSA, 40mM glycine, pH 7.4；And
F) it centrifuges and collects the magnetic particle for being coated with monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510706465.3A CN105403693B (en) | 2015-10-27 | 2015-10-27 | A kind of preparation method of magnetic microparticle chemiluminescence reagent |
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