CN105403693A - Preparation method of magnetic particle chemiluminescence reagent - Google Patents
Preparation method of magnetic particle chemiluminescence reagent Download PDFInfo
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- CN105403693A CN105403693A CN201510706465.3A CN201510706465A CN105403693A CN 105403693 A CN105403693 A CN 105403693A CN 201510706465 A CN201510706465 A CN 201510706465A CN 105403693 A CN105403693 A CN 105403693A
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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
The invention relates to a preparation method of a magnetic particle chemiluminescence reagent, and also relates to a detection kit of human myoglobins. The method comprises the following steps: mixing magnetic particles with a cross-linking agent, and combining the obtained mixture with an antibody to be coated. A polymer is adopted by the method in the invention to close the magnetic particles. The detection regent prepared through the method combines respective advantages of chemiluminescence and a magnetic particle technology, so the magnetic particles make the kit have high specificity and sensitivity, large detection range and fast detection time.
Description
Technical field
The disclosure belongs to immunological technique detection field, particularly relates to a kind of preparation method of magnetic microparticle chemiluminescence kit, and the kit adopting the method to prepare; The disclosure also relates to a kind of quantification kit detecting myoglobins in human serum.
Background technology
Myoglobins is a kind of protein being present in cardiac muscle and Skeletal Muscle Cell matter, and molecular weight is 17.8kD.
After myocyte is impaired, myoglobins is discharged in the circulation system fast.The myoglobins measured in serum is success one of important indicator pouring into diagnosis again after diagnosing acute miocardial infarction (AMI), again infraction and thromboembolism treatment.Concentration is about myoglobin concentration rising in two hours after symptom occurs, therefore can be used as the early stage index of diagnosing cardiac infarction.Pour into remedy measures again according to different, after infraction formation starts, the myoglobin concentration in blood circulation can peak after infraction occurs for 4 to 12 hours, drops to normal level after about 24 hours.Skeletal muscle impaired after and renal function serious hindrance myoglobin concentration also can be caused to raise.
When not having Skeletal muscle injury, myoglobins is used as the early sign of miocardial infarction illness.The negative prediction result of myoglobins is 99%, is used for getting rid of non-Patients With Myocardial Infarction.When applying together in conjunction with other cardiac marker (as CK-MB or Troponin I), myoglobins has remarkable diagnostic value, can be used for assessing potential Acute Myocardial Infarction Patients.
The myoglobins detection means adopted clinically at present comprises: ELISA method and immunoturbidimetry.But the shortcoming of these methods is that insufficient sensitivity is high, detection duration, and the range of linearity is wide not, sample has to carry out dilution process.
In view of above-mentioned weak point, be developed a kind of new detection method, it combines the double dominant of magnetic separation technique and chemiluminescence, such as, disclose a kind of myoglobins quantitative determination reagent kit and detection method thereof in Chinese patent CN102435752B, this kit comprises the anti-myoglobins monoclonal antibody of magnetic microsphere and the alkali phosphatase enzyme mark being marked with anti-myoglobins monoclonal antibody.Change method chemiluminescence is combined with immune magnetic particle, thus provide a kind of reaction system close to homogeneous phase, there is higher detection sensitivity and specificity.But, in prior art antibody bag by the method for magnetic particle be first magnetic particle and antibody are mixed after add crosslinking chemical (as EDC).Even if in CN102435752B, be also that antibody and crosslinking chemical disuccinimidyl suberate are mixed in advance.In these method for coating, crosslinking chemical can damage the activity of antibody.Further, the reagent range of linearity is broad not, and high level sample needs to carry out dilution process in advance.Given this, those skilled in the art wishes to obtain a kind of novel magnetic particle method for coating to improve the defect in above-mentioned technology very much.
Summary of the invention
According to the one side of the application, provide a kind of method for coating of magnetic particle, it comprises step:
A) magnetic particle is contacted with binding buffer liquid, obtain the first potpourri;
B) carbodiimide is contacted 10min to 1h at 20 DEG C to 30 DEG C with the first potpourri, obtain the second potpourri; And
C) monoclonal antibody and the second potpourri are reacted 2h to 4h 20 DEG C to 30 DEG C lucifuges, obtain the magnetic particle being coated with monoclonal antibody.
According to some embodiments, the pH of described binding buffer liquid is 4.5 to 6.0; Preferably, pH is selected from: 4.5,4.8,5.0,5.2,5.5 and above-mentioned any two numerical value between scope.In preferred embodiment, the pH of described binding buffer liquid is 5.0.In a particular embodiment, described binding buffer liquid is MES damping fluid.In preferred embodiment, binding buffer liquid to be pH be 5.0 0.1MMES damping fluid.
According to some embodiments, the particle diameter of described magnetic particle is 1 μm to 3 μm.In a preferred embodiment, the particle diameter of described magnetic particle is 0.8 to 1.2 μm.Technician understands, and in fact magnetic particle used in disclosure method can not be single magnetic particle, but numerous magnetic particle.Therefore, the particle diameter of magnetic particle is the distribution of a particle size range on statistical significance.Such as, when mentioning that particle diameter is the magnetic particle of 1 μm, and do not mean that the particle diameter of each single magnetic particle is just 1 μm, but allow near 1 μm (error of such as ± 10%) scope within.Given this, in the context of this application, the particle diameter of magnetic particle is represented with the scope of particle diameter.
According to some embodiments, in step b) in carbodiimide is contacted 20min to 40min at 20 DEG C to 30 DEG C with the first potpourri, obtain the second potpourri.In a preferred embodiment, step b) in by carbodiimide and the first potpourri at 22 DEG C to 28 DEG C mixing 25min to 35min.In a particular embodiment, the concentration of carbodiimide is 8mg/ml to 15mg/ml, preferred 10mg/ml.
According to some embodiments, step c) in by monoclonal antibody and the second potpourri at 22 DEG C to 28 DEG C lucifuges reaction 3h.In a particular embodiment, monoclonal antibody is myoglobins monoclonal antibody.But technician understands, although have employed specific monoclonal antibody in the instantiation of the application, the antibody of other type any is equally applicable to the application.Because what the method for the application related to is a kind of magnetic particle bag of improvement, by step and closed step, as long as antibody can be bonded to magnetic particle, is not limited to the ad hoc structure (sequence as CDR) of antibody.
According to some embodiments, the method for coating of the magnetic particle of the application also comprises steps d): add Biolipidure to described being coated with in the magnetic particle of monoclonal antibody, carry out 2 to 3h capping at 22 to 28 DEG C; Afterwards at 37 DEG C of reaction 15min to 60min.Optionally, the method for coating of the magnetic particle of the application also comprises step e): stop steps d) in capping.Optionally, the method for coating of the magnetic particle of the application also comprises step f): centrifugal and collection is coated with the magnetic particle of monoclonal antibody.
it is the polymkeric substance (the people J.Biomed.Mater.Res. such as K.Ishihara, 39,323,1998 years) that one has phosphatide polar group 2-methacryl phosphocholine (MPC).
include but not limited to Biolipidure-103, Biolipidure-203, Biolipidure-206, Biolipidure-405, Biolipidure-502, Biolipidure-702, Biolipidure-802, Biolipidure-1002, Biolipidure-1201 and Biolipidure-1301.In the context of this application, Ren Hehe
equivalent product may be used for the method for the application equally.In a particular embodiment, described in
biolipidure-502 or Biolipidure-1002; Preferred Biolipidure-1002.
According to some preferred embodiment, in steps d) in, add Biolipidure to described being coated with in the magnetic particle of monoclonal antibody, carry out 2 to 3h capping at 22 to 28 DEG C, afterwards at 37 DEG C of reactions 15min, 30min or 60min, most preferably react 30min.
The embodiment concrete according to some, stops capping by adding quenching buffers at 22 to 28 DEG C of lucifuge reaction 0.5 to 1.5h.Quenching buffers well known in the art can use.Such as, in a particular embodiment, quenching buffers comprises 10mMPBS, 1g/100mlBSA, 40mM glycocoll, and pH is 7.4.
According to a concrete embodiment, the method for coating of magnetic particle comprises step:
A) 2 μm of magnetic particles are contacted with the 0.1MMES damping fluid of pH5.0, obtain the first potpourri;
B) 10mg/ml carbodiimide is contacted 30min at 20 DEG C to 30 DEG C with the first potpourri, obtain the second potpourri;
C) monoclonal antibody and the second potpourri are reacted 3h 20 DEG C to 30 DEG C lucifuges, obtain the magnetic particle being coated with monoclonal antibody;
D) add Biolipidure-1002 to described being coated with in the magnetic particle of monoclonal antibody, carry out 3h capping at 22 to 28 DEG C, afterwards at 37 DEG C of reaction 30min;
E) add quenching buffers at 20 DEG C to 30 DEG C lucifuge reaction 1h to stop described capping, described quenching buffers comprises 10mMPBS, 1g/100mlBSA, 40mM glycocoll, and pH is 7.4;
F) centrifugal and collection is coated with the magnetic particle of monoclonal antibody.
According to the another aspect of the application, provide a kind of magnetic particle being coated with monoclonal antibody, it is prepared by the method for the application.
According to the another aspect of the application, additionally provide the purposes of the magnetic particle being coated with monoclonal antibody in preparation detection reagent prepared by the method for the application.
According to the another aspect of the application, additionally provide a kind of detection reagent, it comprises the magnetic particle being coated with monoclonal antibody of the application.
According to the another aspect of the application, additionally provide a kind of myoglobin assay kit, it comprises or by forming as follows: Magneto separate reagent R1, labelled reagent R2, and substrate solution.In a specific embodiment, Magneto separate reagent R1 comprises the magnetic particle being coated with myoglobins monoclonal antibody.In R1, the magnetic particle being coated with myoglobins monoclonal antibody allows to be placed in suitable damping fluid.The damping fluid of R1 preferably comprises: 0.6g/100mlNaCl, 0.2g/100ml polysorbas20,0.2g/100mlNaN3,50mMTris-HCl damping fluid, pH7.6.The concentration of described myoglobins monoclonal antibody in R1 is 2mg/ml.In a specific embodiment, labelled reagent R2 comprises the myoglobins monoclonal antibody of enzyme labeling.In preferred embodiment, labelled reagent R2 comprises myoglobins monoclonal antibody and the damping fluid of alkali phosphatase enzyme mark.Damping fluid preferably comprises: 0.6g/100mlNaCl, 0.2g/100ml polysorbas20,0.2g/100mlNaN3,50mMTris-HCl damping fluid, pH7.6.The concentration of myoglobins monoclonal antibody in R2 of described alkali phosphatase enzyme mark is 0.1 μ g/ml.
In a specific embodiment, substrate solution comprises the substrate of enzyme, such as chemical luminous substrate.
In other embodiments, the myoglobin assay kit of the application also comprises myoglobins standard items.Standard items can be commercially available acquisitions, also can prepare voluntarily; Wherein comprise the myoglobins of concentration known.Technician is clear, and the standard items object in kit is drafting typical curve.Therefore, any calibration object concentration that can cover sensing range can use.The concentration of myoglobins is selected from but is not limited to one or more as follows: 0,10,50,100,300,800, the human muscle hemoglobin of 1500ng/ml.
In other embodiments, the myoglobin assay kit of the application also comprises washing lotion.Preferably, washing lotion contains polysorbas20, sodium chloride and PC300.
In a particular embodiment, the magnetic particle being coated with myoglobins monoclonal antibody is closed with
include but not limited to Biolipidure-103, Biolipidure-203, Biolipidure-206, Biolipidure-405, Biolipidure-502, Biolipidure-702, Biolipidure-802, Biolipidure-1002, Biolipidure-1201 and Biolipidure-1301.In the context of this application, Ren Hehe
equivalent product may be used for the technical scheme of the application equally.In a particular embodiment, described in
biolipidure-502 or Biolipidure-1002; Preferred Biolipidure-1002.
In a particular embodiment, the magnetic particle being coated with myoglobins monoclonal antibody described in is prepared by the method for coating of the application.
Accompanying drawing explanation
Fig. 1: the comparison of the application's method for coating and contrast method.
◆ method for expressing 1 wrap by after magnetic bead, the magnetic bead amount that each test uses is 20 μ g (y=533.72x+1.7E+5, r
2=0.8352);
■ method for expressing 1 wrap by after magnetic bead, the magnetic bead amount that each test uses is 40 μ g (y=1326.5x+1.8E+5, r
2=0.9656);
▲ method for expressing 2 wrap by after magnetic bead, the magnetic bead amount that each test uses is 10 μ g (y=525.31x+36708, r
2=0.9734).
Fig. 2: the correlativity of the kit of detection kit prepared by the application's method and ripe listing compares.Relationship equation is y=1.0858x-4.8577, r
2=0.9836.
Embodiment
Embodiment
Embodiment 1: contrast method
1. prepare the magnetic particle of myoglobins monoclonal antibody bag quilt
1.1 fully mix magnetic particle (Merck, particle diameter 0.8 to 1.2 μm, article No. 39433087), get 10mg and are placed in test tube, Magneto separate 1 minute, remove supernatant.Add 1ml binding buffer liquid (0.1MMES, pH5.0), after mixing, Magneto separate removed supernatant after 1 minute, mixed magnetic particle after adding 1ml binding buffer liquid.
1.2 add 100 μ g myoglobins monoclonal antibodies, room temperature (22 DEG C to 28 DEG C) mixing 30 minutes.
1.3 add 100 μ l10mg/mlEDC (face with now joining, solvent is the binding buffer liquid of ice) room temperature lucifuge mixes 3 hours.Magneto separate removes supernatant, adds dcq buffer liquid (50mMTBS, pH7.4), and mixing Magneto separate removes supernatant, three times repeatedly.
1.4 add 1ml quenching buffers (10mMPBS, pH7.4, containing 1g/100mlBSA, 40mM glycocoll) room temperature lucifuge mixes 1 hour, and Magneto separate removes supernatant.
1.5 add 1ml magnetic particle storage liquid (100mMPBS, pH7.4, containing 0.1g/100ml Tween 80,1g/100mlBSA), and mixing Magneto separate removes supernatant.Three times repeatedly, be finally placed in storage liquid 4C and save backup.
2.R1 preparation
With dilution (0.6g/100mlNaCl, 0.2g/100ml polysorbas20,0.2g/100mlNaN3,50mMTris-HCl damping fluid, pH7.6), the magnetic particle 1:5 marked is diluted, namely obtain R1 reagent.
Embodiment 2: the magnetic particle method for coating after optimization
1. prepare the magnetic particle of myoglobins monoclonal antibody bag quilt
1.1 fully mix magnetic particle (Merck, particle diameter 0.8 to 1.2 μm, article No. 39433087), get 10mg and are placed in test tube, Magneto separate 1 minute, remove supernatant.Add 1ml binding buffer liquid (0.1MMES, pH5.0), after mixing, Magneto separate removed supernatant after 1 minute, mixed magnetic particle after adding 1ml binding buffer liquid.
1.2 add 100 μ l10mg/mlEDC (face with now joining, solvent is the binding buffer liquid of ice), room temperature (at 22 DEG C to 28 DEG C) mixing 30 minutes.
1.3 add 100 μ g myoglobins monoclonal antibodies, and room temperature lucifuge mixes 3 hours.Magneto separate removes supernatant, adds dcq buffer liquid (50mMTBS, pH7.4), and mixing Magneto separate removes supernatant, three times repeatedly.
1.4 add Biolipidure-1002 carries out closed 3h in room temperature, and is placed on 37 DEG C of process 30min.
1.5 add 1ml quenching buffers (10mMPBS, pH7.4, containing 1g/100mlBSA, 40mM glycocoll) room temperature lucifuge mixes 1 hour, and Magneto separate removes supernatant.
1.6 add 1ml magnetic particle storage liquid (100mMPBS, pH7.4, containing 0.1g/100ml Tween 80,1g/100mlBSA), and mixing Magneto separate removes supernatant, three times repeatedly, is finally placed in storage liquid 4C and saves backup.
2.R1 preparation
With dilution (0.6g/100mlNaCl, 0.2g/100ml polysorbas20,0.2g/100mlNaN3,50mMTris-HCl damping fluid, pH7.6), the magnetic particle 1:5 marked is diluted, namely obtain R1 reagent.
3. result
The antibody bag of embodiment 1 is by magnetic particle method (contrast method), and be first magnetic particle and antibody are mixed, then add crosslinking chemical EDC, EDC can damage antibody activity.In order to protect antibody activity, have employed the optimization method of embodiment 2.Test findings (Fig. 1) shows: in the method for embodiment 1, antibody activity is good, but linear high some ratio is limited, need by bag by after magnetic particle consumption bring up to 40 μ g from each test 20 μ g and just can obtain good high some ratio, this illustrates that bag is poor by efficiency.The method general performance of embodiment 2 is better, each test use 10 μ g wrap by after magnetic particle, just can reach better linear effects.
Closing of embodiment 3. magnetic particle
In order to compare the sealing effect of confining liquid, after the step 1.3 of embodiment 2, by bag by after magnetic particle dcq buffer liquid cleaning after be divided into two, add 200 μ lBiolipidure-502 or Biolipidure-1002 respectively and close.
Result (table 1) show Biolipidure-1002 close after magnetic particle in the reaction signal to noise ratio (S/N ratio) be 5.304; Sensitivity is better, and high level linear scaling is also better.Because Biolipidure is the polymkeric substance containing different molecular weight, make magnetic particle sealing effect better.
Magnetic particle after Biolipidure-1002 closes, is placed in 37 DEG C and processes 15min, 30min and 60min respectively.Test findings (table 2) shows the magnetic particle after processing 15min at 37 DEG C, performance in the reaction and undressedly obviously not distinguish.
And the magnetic particle processed at 37 DEG C after 30min, show best specificity and sensitivity in the reaction, signal to noise ratio (S/N ratio) reaches 7.224, and the theoretical concentration of linear high level ratio and standard point is more pressed close to.
Process the magnetic particle after 60min at 37 DEG C, background value raises, and causes signal to noise ratio (S/N ratio) to decline, and the processing time is excessively of a specified duration, and high level ratio is deteriorated.
The comparison of table 1.Biolipidure-502 and Biolipidure-1002 sealing effect
The comparison of table 2.Biolipidure-1002 effect different off-period
The preparation of the alkali phosphatase enzyme mark myoglobins antibody in embodiment 4:R2 reagent
Get 1mg alkaline phosphatase, add 0.5mg myoglobins monoclonal antibody; Mend volume to 500 μ l with the 20mMPBS of pH7.2, put into bag filter, the 20mMPBS being placed in pH7.2 dialyses three times.
Add 10 μ l5% glutaraldehydes after taking-up, room temperature lucifuge mixes 2 hours.The 20mMPBS being again placed in pH7.2 dialyses three times, and removing glutaraldehyde, obtains the myoglobins monoclonal antibody of alkali phosphatase enzyme mark.
Be kept at by the antibody marked in 50mMTris-HCl (containing 0.9g/100mlNaCl, 0.1g/100mlNaN3), add the glycerine of equal volume, packing is put into-20 DEG C and is saved backup.
With dilution (0.6g/100mlNaCl, 0.2g/100ml polysorbas20,0.2g/100mlNaN3,50mMTris-HCl damping fluid, pH7.6), the antibody 1:5000 marked is diluted, namely obtain R2 reagent.
Embodiment 5: the preparation of human muscle hemoglobin standard items
Purchased standard items concentrate (purchased from Fitzgerald) is diluted to working concentration with sheep serum, is respectively 10,50,100,300,800,1500ng/ml.
Embodiment 6: prepared by washing lotion
Washing lotion is Tris-HCl damping fluid, simultaneously also containing concentration be the polysorbas20 of 0.25g/100ml, concentration is the sodium chloride of 0.6g/100ml and the PC300 of 0.1g/100ml.
Embodiment 7: clinical testing and performance index
Each reagent prepared by embodiment 2 and 4 to 6 is assembled into according to kit of the present disclosure.Carry out clinical testing with kit of the present disclosure, serum sample number is 458 examples, from patient and Healthy People.
To have ratified the myoglobins chemical luminescence reagent kit kit in contrast gone on the market abroad; Its Cleaning Principle is double antibody sandwich method; Its kit consists of: reagent 1 comprises the magnetic particle of myoglobins labeling of monoclonal antibody, and reagent 2 comprises the myoglobins monoclonal antibody of acridinium ester label.
First detect with the myoglobins chemical luminescence reagent kit ratifying to go on the market, then use this kit measurement.Result (Fig. 2) shows, the relationship equation of two kit measured values is y=1.0858x-4.8577, R
2=0.9836, illustrate that this kit is better with the measured value consistance of commercialized product.
Kit of the present disclosure realizes following performance index:
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 cholerythrin≤40mg/dl, haemoglobin≤1000mg/dl, during triglyceride≤1000mg/dl, measured value change≤5%;
6) reagent stability preserving 5 days at 37 DEG C is good, and the standard items preserved 10 days at 37 DEG C have good stability;
7) the difference between batch <10% of three batches of reagent of successively preparation.
The advantage of technical scheme of the present disclosure: two monoclonal antibodies are marked on alkaline phosphatase and magnetic particle by kit of the present disclosure respectively, wherein magnetic particle coated antibody is through optimizing, ensure that the bag of better antibody activity and Geng Gao is by efficiency, then utilize polymkeric substance to close magnetic particle and carry out hyperthermic treatment, kit is made to detect myoglobins in human serum in accurate homogeneous reaction, there is higher specificity, sensitivity, larger sensing range and fast reaction time.
Claims (18)
1. a method for coating for magnetic particle, it comprises step:
A) magnetic particle is contacted with binding buffer liquid, obtain the first potpourri;
B) carbodiimide is contacted 10min to 1h at 20 DEG C to 30 DEG C with the first potpourri, obtain the second potpourri; And
C) monoclonal antibody and the second potpourri are reacted 2h to 4h 20 DEG C to 30 DEG C lucifuges, obtain the magnetic particle being coated with monoclonal antibody.
2. method according to claim 1, the pH of wherein said binding buffer liquid is 4.5 to 6.0; Preferably, pH is selected from: 4.5,4.8,5.0,5.2,5.5 and above-mentioned any two numerical value between scope; More preferably, described pH is 5.0;
Described binding buffer liquid is MES damping fluid;
More preferably, described binding buffer liquid to be pH be 5.0 0.1MMES damping fluid.
3. method according to claim 1, the particle diameter of wherein said magnetic particle is 1 μm to 3 μm; The particle size range of preferred described magnetic particle is within 0.8 μm to 1.2 μm.
4. method according to claim 1, wherein
In step b) in carbodiimide is contacted 20min to 40min at 20 DEG C to 30 DEG C with the first potpourri; Preferably, step b) in by carbodiimide and the first potpourri at 22 DEG C to 28 DEG C mixing 25min to 35min.
5. method according to claim 1, wherein step b) concentration of carbodiimide is 8 to 15mg/ml, preferred 10mg/ml.
6. method according to claim 1, wherein step c) in by monoclonal antibody and the second potpourri at 22 DEG C to 28 DEG C lucifuges reaction 3h.
7. method according to claim 1, described monoclonal antibody is myoglobins monoclonal antibody.
8. method according to claim 1, described method also comprises step:
D) add Biolipidure to described being coated with in the magnetic particle of monoclonal antibody, carry out 2 to 3h capping at 22 to 28 DEG C, afterwards at 37 DEG C of reaction 15 to 60min, preferably at 37 DEG C of reaction 30min; And
E) optionally, described capping is stopped;
F) optionally, centrifugal and collection is coated with the magnetic particle of monoclonal antibody;
Described Biolipidure is Biolipidure-502 or Biolipidure-1002; Preferred Biolipidure-1002.
9. method according to claim 8, carries out described step e by adding quenching buffers at 22 to 28 DEG C of lucifuge reaction 0.5 to 1.5h); Preferably, described quenching buffers comprises 10mMPBS, 1g/100mlBSA, 40mM glycocoll, and pH is 7.4.
10. the method according to any one of claim 1-9, comprises step:
A) magnetic particle of 0.8 to 1.2 μm is contacted with the 0.1MMES damping fluid of pH5.0, obtain the first potpourri;
B) 10mg/ml carbodiimide is contacted 30min at 20 DEG C to 30 DEG C with the first potpourri, obtain the second potpourri;
C) monoclonal antibody and the second potpourri are reacted 3h 20 DEG C to 30 DEG C lucifuges, obtain the magnetic particle being coated with monoclonal antibody;
D) add Biolipidure-1002 to described being coated with in the magnetic particle of monoclonal antibody, carry out 3h capping at 22 to 28 DEG C, afterwards at 37 DEG C of reaction 30min;
E) add quenching buffers at 20 DEG C to 30 DEG C lucifuge reaction 1h to stop described capping, described quenching buffers comprises 10mMPBS, 1g/100mlBSA, 40mM glycocoll, and pH is 7.4; And
F) centrifugal and collection is coated with the magnetic particle of monoclonal antibody.
11. 1 kinds of magnetic particles being coated with monoclonal antibody, it is prepared by the method described in any one of claim 1-10.
The purposes of magnetic particle in preparation detection reagent of monoclonal antibody is coated with 12. according to claim 11.
13. detect a reagent, it comprises the magnetic particle being coated with monoclonal antibody according to claim 11.
14. a myoglobin assay kit, it comprises or by forming as follows:
Magneto separate reagent R1, labelled reagent R2, and substrate solution;
Wherein said Magneto separate reagent R1 comprises: the magnetic particle and the damping fluid that are coated with myoglobins monoclonal antibody;
Described labelled reagent R2 comprises: the myoglobins monoclonal antibody of enzyme labeling and damping fluid;
Described substrate solution comprises: chemical luminous substrate,
Preferably, described damping fluid comprises 0.6g/100mlNaCl, 0.2g/100ml polysorbas20,0.2g/100mlNaN3,50mMTris-HCl damping fluid, and pH is 7.6;
Preferably, described enzyme is alkaline phosphatase.
15. myoglobin assay kits according to claim 14, it also comprises myoglobins standard items;
Preferably, the concentration of myoglobins standard items is selected from one or more as follows: 0,10,50,100,300,800, the human muscle hemoglobin of 1500ng/ml.
16. myoglobin assay kits according to claims 14 or 15, it also comprises washing lotion; Preferably described washing lotion contains polysorbas20, sodium chloride and PC300.
17. myoglobin assay kit according to claim 14, the wherein said magnetic particle being coated with myoglobins monoclonal antibody is closed with Biolipidure; Preferably, described Biolipidure is Biolipidure-502 or Biolipidure-1002; More preferably described Biolipidure is Biolipidure-1002.
18. myoglobin assay kits according to claim 14, the wherein said magnetic particle being coated with myoglobins monoclonal antibody is prepared by the method described in any one of claim 1-10.
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| CN107655880A (en) * | 2017-09-21 | 2018-02-02 | 苏州新波生物技术有限公司 | A kind of myoglobins magnetic microparticle chemiluminescence detection kit and its application method |
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| CN107462708A (en) * | 2017-06-28 | 2017-12-12 | 迈克生物股份有限公司 | Method, application and the kit of antigen or antibody coating magnetic particle |
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| CN107655880A (en) * | 2017-09-21 | 2018-02-02 | 苏州新波生物技术有限公司 | A kind of myoglobins magnetic microparticle chemiluminescence detection kit and its application method |
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| CN108828233A (en) * | 2018-08-11 | 2018-11-16 | 金华市强盛生物科技有限公司 | A kind of myoglobin assay kit and preparation method thereof |
| CN109765382A (en) * | 2018-12-12 | 2019-05-17 | 北京九强生物技术股份有限公司 | A kind of latex enhancing immune of serum cardiac troponin T is than turbid detection kit |
| CN109765382B (en) * | 2018-12-12 | 2021-10-08 | 北京九强生物技术股份有限公司 | Latex-enhanced immunoturbidimetric assay kit for cardiac troponin T |
| CN111007254A (en) * | 2019-11-15 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Protein-coated amino microsphere, preparation method thereof and angiotensin I detection kit |
| CN113125721A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous detection kit for creatine kinase isoenzyme and application thereof |
| CN113125700A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Homogeneous detection kit for creatine kinase isoenzyme and application thereof |
| CN113125721B (en) * | 2019-12-31 | 2023-07-07 | 科美博阳诊断技术(上海)有限公司 | Homogeneous detection kit for creatine kinase isozymes and application thereof |
| CN113125700B (en) * | 2019-12-31 | 2023-08-08 | 科美博阳诊断技术(上海)有限公司 | Homogeneous detection kit for creatine kinase isozymes and application thereof |
| CN113311174A (en) * | 2021-05-14 | 2021-08-27 | 宁波瑞源生物科技有限公司 | Myoglobin antibody-enzyme marker and preparation and application thereof |
| CN113311174B (en) * | 2021-05-14 | 2022-05-13 | 宁波瑞源生物科技有限公司 | Myoglobin antibody-enzyme marker and preparation and application thereof |
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