CN107462708A - Method, application and the kit of antigen or antibody coating magnetic particle - Google Patents
Method, application and the kit of antigen or antibody coating magnetic particle Download PDFInfo
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- CN107462708A CN107462708A CN201710507484.2A CN201710507484A CN107462708A CN 107462708 A CN107462708 A CN 107462708A CN 201710507484 A CN201710507484 A CN 201710507484A CN 107462708 A CN107462708 A CN 107462708A
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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
Abstract
The present invention relates to the method for antigen or antibody coating magnetic particle, and application of the magnetic particle prepared by this method in chemiluminescence immune assay.The invention further relates to a kind of kit, including antigen or the coated magnetic particle of antibody.The method of antigen or antibody provided by the invention coating magnetic particle, the characteristics of there is antigen or antibody detected signal value is high after magnetic particle in coating, stability is good.
Description
Technical field
The invention belongs to field of biological detection, and in particular to the method for antigen or antibody coating magnetic particle.
Background technology
Chemiluminescence immunoassay is improved on the basis of two methods of radiommunoassay and ELISA
, the advantages of it combines above two method：High sensitivity, the range of linearity is wide, simple to operate, as a result stablizes, repeatability
It is good.Therefore, chemiluminescence immunoassay is the outstanding person in current external diagnosis reagent detection method.This method will be by that will resist
Former or antibody is fixed on certain special carrier, using the specific reaction of antigen-antibody, and relies on direct or indirect shiner
Matter, tested substance concentration is converted into optical signalling and reflected.
Magnetic particle is as carrier the most frequently used in chemiluminescence immune assay, suitable for most vitro detection projects,
It is combined by the carboxyl and the amino of envelope antigen or antibody on magnetic particle surface, reaches the effect of immobilized antigen or antibody.Mesh
Before, the method for the conventional envelope antigen of magnetic particle or antibody is divided into one-step method and two-step method.
Publication No. CN106404757A Chinese patent discloses a kind of nanometer magnetic bead preparation side of RA33 antigen coats
Method, the patent use one-step method, that is, are coated with process and add EDC, magnetic particle and antigen in solution simultaneously and react.One-step method operates
Step is easy, time-consuming less, but examines the problem of not measuring signal value or too low signal value after some antigen coats being present.
Publication No. CN101949943A Chinese patent discloses a kind of magnetic of antithyrotropic hormone monoclonal antibody
The preparation method of micronised suspensions, the patent use two-step method, i.e. magnetic particle is first activated with EDC, is added after standing and is waited to be coated with
Antibody.In two-step method, EDC has only activated the carboxyl on magnetic particle, the carboxyl specific aim after activation and treat coated antigen or
The amino of antibody combines, and efficiency is higher, and last detected signal value also accordingly improves.But in experimentation it was found that certain
After a little antigens or antibody are coated on magnetic particle, its less stable, the calibration cycle and the term of validity of kit have impact on.
Therefore, when being coated with magnetic particle using antigen or antibody, it is necessary to a kind of method both can guarantee that its was coated with efficiency, it is made
Final detected signal value is higher, ensures the stability of antigen or antibody in coating after magnetic particle again.
The content of the invention
The present invention is to overcome above-mentioned problem of the prior art, there is provided a kind of method of antigen or antibody coating magnetic particle, bag
Include following steps：
Step 1：Magnetic particle is taken, magnetic particle is resuspended with buffer A；
Step 2：Activating solution is taken, is added in the magnetic particle solution that step 1 has been resuspended, is mixed；
Step 3：Antigen or antibody are added in the magnetic particle solution that step 2 has activated, mixed；
Step 4：Activating solution is taken, is added in step 3 solution, is mixed；
Step 5：Confining liquid is taken, is added in step 4 solution, is mixed；
Step 6：Buffer B is taken, the magnetic particle of cleaning step five, completes antigen or antibody coating magnetic particle.
Further：
Step 2: activating solution described in four is EDC.HCL or DCC solution；
Buffer A described in step 1 is MES solution；
Confining liquid described in step 5 is the one or more of Tris or BSA or casein solution；
Buffer B described in step 6 is TBST or PBS or the PBS solution containing Tween.
Further：
Magnetic particle concentration after the resuspension magnetic particle of buffer A described in step 1 is 2mg/ml~20mg/ml；
EDC.HCL or DCC and magnetic particle mass ratio are 1 in activating solution described in step 2:20~5:1；
Antigen described in step 3 or antibody and magnetic particle mass ratio are 1:2000~1:50；
EDC.HCL or DCC and magnetic particle mass ratio are 1 in activating solution described in step 4:20~5:1；
Magnetic particle in step 1 had been cleaned before being resuspended by buffer A by buffer A；
Above-mentioned magnetic particle average grain diameter is 1 μm~3 μm；
Above-mentioned magnetic particle quality is the magnetic particle quality before being cleaned in step 1 by buffer A.
Magnetic particle prepared by the method for antigen or antibody coating magnetic particle provided by the invention is applied in chemiluminescence immunoassay
In analysis, the detected signal value of antigen or antibody in coating after magnetic particle, the chemiluminescence immune assay bag can be improved
Include the chemiluminescence immunoassay point for various specific total antibodies, specific IgG, IgM antibody-likes, specific antigen in human serum
Analysis.
Further：The chemiluminescence immune assay include RNP antigens, SCL-70 antigens, JO-1 antigens, HIV antigens,
The chemiluminescence immune assay of at least one of HCV antigens, HBeAg antibody, insulin, FT3, FT4 and Renin.
The present invention also provides a kind of kit, and the kit includes antigen or the coated magnetic particle of antibody, the antigen
Or the coated magnetic particle of antibody is prepared by the method for antigen of the present invention or antibody coating magnetic particle.Antigen provided by the invention
Or the method for antibody coating magnetic particle, there is advantages below：
1st, detected signal value is high after magnetic particle in coating for antigen or antibody；
2nd, antigen or antibody magnetic particle rear stability in coating are good.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
It is not used in limitation the scope of the present invention.
It is as follows to be related to each solution compound method in the method for antigen or antibody coating magnetic particle provided by the invention：
1st, 0.1mol/L MES solutions are prepared：Weigh 19.52g MES to be dissolved in 1L purified waters, pH value is adjusted to 6.0 with NaOH
±0.2。
2nd, 10mg/ml EDC.HCL solution is prepared：The EDC.HCL for weighing 10mg is dissolved in the cold MES solutions of 1ml.
3rd, 10mg/ml DCC solution is prepared：The 10mg/ml DCC for weighing 10mg are dissolved in 1ml DMSO solutions.
4th, 1mol/L Tris solution is prepared：121.1g Tris are weighed to be dissolved in 1L purified waters.
5th, 10%BSA solution is prepared：10g BSA are weighed to be dissolved in 100ml purified waters.
6th, 10% casein solution is prepared：10g caseins are weighed to be dissolved in 100ml purified waters.
7th, TBST solution is prepared：Weigh 3.03g Tris to be dissolved in 1L purified waters, then 8.775g is added into above-mentioned solution
NaCl, pH value is adjusted to 7.3~7.4 with HCL solution.
It is related to material in the method for antigen or antibody coating magnetic particle provided by the invention and reagent source is as follows：
Magnetic particle (Sichuan mikey biology new material technology Co., Ltd LOT:XCL1137)
MES (Suzhou subfamily Cat：M0006)
EDC.HCL (Suzhou subfamily Cat：E0009)
DCC(Sigma Cat：BCBM3570V)
Tris(ANGUS)
BSA(BOVOGEN Cat：BSAS 1.0)
Casein (Sigma Cat：SLBF5266V)
NaCl (analysis of Chengdu Cologne Chemical Company is pure)
NaOH (Guangdong Guanghua Science and Technology Co., Ltd.'s analysis is pure)
DMSO (Chengdu Chang Lian chemical reagents Co., Ltd chemistry is pure)
HCL (analysis of Chengdu Cologne Chemical Company is pure)
RNP antigens (Sichuan mikey biology new material technology Co., Ltd Cat:XCL1104)
SCL-70 antigens (Sichuan mikey biology new material technology Co., Ltd Cat:XCL1105)
Anti- (the Sichuan mikey biology new material technology Co., Ltd Cat of HBeAg monoclonal antibodies -1:XCL1006)
HRP- anti-human IgG antibodies (Sichuan mikey biology new material technology Co., Ltd Cat:XCL1158)
Anti-RNP Control(Bio-Rad Laboratories Cat:LiquichekTMAutoimmune
Controls116)
Anti-Scl-70 Control(Bio-Rad Laboratories Cat:LiquichekTMAutoimmune
Controls117)
(the mikey Biological Co., Ltd. Cat of Sample dilution 4:IM4212464)
HRP-HBeAb monoclonal antibodies (Beijing Key-Biotechnology Co., Ltd Cat:B311H005)
Recombinant hepatitis b virus e antigens (Sichuan mikey biology new material technology Co., Ltd Cat:XCL1367)
In the examples below, by taking tri- projects of RNP, SCL-70 and HBeAg as an example, one-step method, two-step method is respectively adopted
Magnetic particle, and the respective other components of kit of supporting three projects are coated with the inventive method, detect respective items purpose master respectively
Calibration object.Compare the detected signal value of one-step method, two-step method and the inventive method coating magnetic particle, then detected signal value is relative
After higher 37 DEG C of water-baths of magnetic particle, compare the signal retention rate of magnetic particle, in of the invention, magnetic particle signal is protected after 37 DEG C of water-baths
Rate is stayed to be defined as follows：
Signal retention rate=(37 DEG C of water-baths N days after magnetic particle detected signal value/magnetic particle be just coated with detected signal value) *
100%
Embodiment 1
One-step method, two-step method and the inventive method is respectively adopted by RNP antigen coat magnetic particles, supporting HRP- anti-human igg
Antibody, the main calibration object CAL1-CAL6 of RNP projects is detected respectively.The higher antigen magnetic particle of detected signal value is positioned over 37 DEG C
After water-bath 7 days, 14 days, then supporting HRP- anti-human IgG antibodies, the main calibration object CAL1-CAL6 of RNP projects is detected respectively.Calculate 37
After DEG C water-bath 7 days, 14 days, antigen magnetic particle signal retention rate.
The main calibration object preparation method of RNP projects is as follows：
CAL1:Sample dilution 4
CAL2:Anti-RNP Control high level point is diluted 4096 times by Sample dilution 4
CAL3:Anti-RNP Control high level point is diluted 512 times by Sample dilution 4
CAL4:Anti-RNP Control high level point is diluted 128 times by Sample dilution 4
CAL5:Anti-RNP Control high level point is diluted 32 times by Sample dilution 4
CAL6:Anti-RNP Control high level point is diluted 16 times by Sample dilution 4
Antigen coat magnetic particle preparation method is as follows：
One-step method is coated with RNP magnetic particles：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 10mg/ml to make its concentration；
2nd, activation coating：EDC.HCL solution is matched somebody with somebody in enchashment, by EDC.HCL and magnetic particle mass ratio 1:20 ratio is to resuspension
EDC.HCL solution is added in good magnetic particle, is vortexed and mixes；It is again 1 by antigen and magnetic particle mass ratio:2000 coating ratio
Being added in the magnetic particle good to above-mentioned activation needs coated RNP antigens, is vortexed after mixing, and room temperature is horizontal to be mixed 12 hours；
3rd, close：1mol/L Tris solution is added into above-mentioned magnetic particle, room temperature is horizontal to be mixed 3 hours；
4th, clean：Above-mentioned magnetic particle is cleaned with TBST solution 5 times, completes RNP antigen coat magnetic particles.
Two-step method is coated with RNP magnetic particles：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 10mg/ml to make its concentration；
2nd, activate：EDC.HCL solution is matched somebody with somebody in enchashment, by EDC.HCL and magnetic particle mass ratio 1:20 ratio is to being resuspended
EDC.HCL solution is added in magnetic particle, is vortexed after mixing, room temperature is horizontal to be mixed 30 minutes；
3rd, it is coated with：It is 1 by antigen and magnetic particle mass ratio:2000 coating ratio adds into the good magnetic particle of above-mentioned activation
Enter to need coated RNP antigens, be vortexed after mixing, room temperature is horizontal to be mixed 12 hours；
4th, close：1mol/L Tris solution is added into above-mentioned magnetic particle, room temperature is horizontal to be mixed 3 hours；
5th, clean：Above-mentioned magnetic particle is cleaned with TBST solution 5 times, completes RNP antigen coat magnetic particles.
The inventive method is coated with RNP magnetic particles：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 10mg/ml to make its concentration；
2nd, activate：EDC.HCL solution is matched somebody with somebody in enchashment, by EDC.HCL and magnetic particle mass ratio 1:20 ratio is to being resuspended
EDC.HCL solution is added in magnetic particle, is vortexed after mixing, room temperature is horizontal to be mixed 30 minutes；
3rd, it is coated with：It is 1 by antigen and magnetic particle mass ratio:2000 coating ratio adds into the good magnetic particle of above-mentioned activation
Enter to need coated RNP antigens, be vortexed after mixing, room temperature is horizontal to be mixed 12 hours；
4th, it is re-activated：EDC.HCL solution is matched somebody with somebody in enchashment, by EDC.HCL and magnetic particle mass ratio 1:20 ratio is to being coated with
EDC.HCL solution is added in the magnetic particle of RNP antigens again, is vortexed after mixing, room temperature is horizontal to be mixed 2 hours；
5th, close：1mol/L Tris solution is added into above-mentioned magnetic particle, room temperature is horizontal to be mixed 3 hours；
6th, clean：Above-mentioned magnetic particle is cleaned with TBST solution 5 times, completes RNP antigen coat magnetic particles.
1 is shown in Table using signal retention rate after the above-mentioned three kinds RNP antigen magnetic particle signal values for being coated with technique and 37 DEG C of water-baths.
Table 1
Result above shows, magnetic particle is coated with using one-step method, its RNP antigen magnetic particle detected signal value compared with two-step method and
The inventive method is low, therefore does not carry out 37 DEG C of water-baths to one-step method coating magnetic particle.And by RNP antigen magnetic particle detected signal values
After higher two-step method and the antigen magnetic particle of the inventive method are positioned over 37 DEG C of water-baths 7 days, 14 days, the inventive method RNP resists
Former magnetic particle signal retention rate is compared with two-step method height.
Embodiment 2
One-step method, two-step method and above-mentioned optimize technique is respectively adopted to resist SCL-70 antigen coat magnetic particles, supporting HRP-
Human IgG antibody, the main calibration object CAL1-CAL6 of detection SCL-70 projects.The higher antigen magnetic particle of detected signal value is positioned over
After 37 DEG C of water-baths 7 days, 14 days, then supporting HRP- anti-human IgG antibodies, the main calibration object CAL1-CAL6 of detection SCL-70 projects.Calculate
After 37 DEG C of water-baths 7 days, 14 days, antigen magnetic particle signal retention rate.
Antigen coat magnetic particle preparation method is as follows：
The main calibration object preparation method of SCL-70 projects is as follows：
CAL1:Sample dilution 4
CAL2:Anti-Scl-70 Control high level point is diluted 4096 times by Sample dilution 4
CAL3:Anti-Scl-70 Control high level point is diluted 512 times by Sample dilution 4
CAL4:Anti-Scl-70 Control high level point is diluted 256 times by Sample dilution 4
CAL5:Anti-Scl-70 Control high level point is diluted 64 times by Sample dilution 4
CAL6:Anti-Scl-70 Control high level point is diluted 32 times by Sample dilution 4
One-step method is coated with SCL-70 magnetic particles：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 2mg/ml to make its concentration；
2nd, activation coating：DCC solution is matched somebody with somebody in enchashment, by DCC and magnetic particle mass ratio 1:1 ratio is to the magnetic particle being resuspended
Middle addition DCC solution, it is vortexed and mixes；It is again 1 by antigen and magnetic particle mass ratio:200 coating ratio is good to above-mentioned activation
Being added in magnetic particle needs coated SCL-70 antigens, is vortexed after mixing, and room temperature is horizontal to be mixed 12 hours；
3rd, close：It is molten that 1mol/L Tris solution, 10%BSA solution and 10% casein are added into above-mentioned magnetic particle
The mixing confining liquid of liquid, room temperature is horizontal to be mixed 3 hours；
4th, clean：Above-mentioned magnetic particle is cleaned with PBS solution 5 times, completes SCL-70 antigen coat magnetic particles.
Two-step method is coated with SCL-70 magnetic particles：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 2mg/ml to make its concentration；
2nd, activate：DCC solution is matched somebody with somebody in enchashment, by DCC and magnetic particle mass ratio 1:1 ratio adds into the magnetic particle being resuspended
Enter DCC solution, be vortexed after mixing, room temperature is horizontal to be mixed 30 minutes；
3rd, it is coated with：It is 1 by antigen and magnetic particle mass ratio:200 coating ratio adds into the good magnetic particle of above-mentioned activation
Enter to need coated SCL-70 antigens, be vortexed after mixing, room temperature is horizontal to be mixed 12 hours；
4th, close：It is molten that 1mol/L Tris solution, 10%BSA solution and 10% casein are added into above-mentioned magnetic particle
The mixing confining liquid of liquid, room temperature is horizontal to be mixed 3 hours；
5th, clean：Above-mentioned magnetic particle is cleaned with PBS solution 5 times, completes SCL-70 antigen coat magnetic particles.
The inventive method is coated with SCL-70 magnetic particles：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 20mg/ml to make its concentration；
2nd, activate：DCC solution is matched somebody with somebody in enchashment, by DCC and magnetic particle mass ratio 1:1 ratio adds into the magnetic particle being resuspended
Enter DCC solution, be vortexed after mixing, room temperature is horizontal to be mixed 30 minutes；
3rd, it is coated with：It is 1 by antigen and magnetic particle mass ratio:200 coating ratio adds into the good magnetic particle of above-mentioned activation
Enter to need coated SCL-70 antigens, be vortexed after mixing, room temperature is horizontal to be mixed 12 hours；
4th, it is re-activated：DCC solution is matched somebody with somebody in enchashment, by DCC and magnetic particle mass ratio 1:1 ratio is to being coated with SCL-70 antigens
Magnetic particle in add DCC solution again, be vortexed after mixing, room temperature is horizontal to be mixed 2 hours；
5th, close：It is molten that 1mol/L Tris solution, 10%BSA solution and 10% casein are added into above-mentioned magnetic particle
The mixing confining liquid of liquid, room temperature is horizontal to be mixed 3 hours；
6th, clean：Above-mentioned magnetic particle is cleaned with PBS solution 5 times, completes SCL-70 antigen coat magnetic particles.
Seen using signal retention rate after the above-mentioned three kinds SCL-70 antigen magnetic particle signal values for being coated with technique and 37 DEG C of water-baths
Table 2.
Table 2
Result above shows, is coated with magnetic particle using one-step method, its SCL-70 antigen magnetic particle detected signal value is compared with two steps
Method and the inventive method are low, therefore do not carry out 37 DEG C of water-baths to one-step method coating magnetic particle.And SCL-70 antigens magnetic particle is examined
Survey the higher two-step method of signal value and the inventive method antigen magnetic particle be positioned over 37 DEG C of water-baths 7 days, 14 days after, present invention side
Method SCL-70 antigen magnetic particle signal retention rates are compared with two-step method height.
Embodiment 3
One-step method, two-step method and above-mentioned optimize technique is respectively adopted anti-HBeAg monoclonal antibodies -1 are coated with magnetic particle, match somebody with somebody
Cover HRP-HBeAb monoclonal antibodies, the main calibration object CAL1-CAL7 of detection recombinant hepatitis b virus e antigens.Detected signal value is higher
After magnetic particle is positioned over 37 degree of water-baths 7 days, 14 days, then supporting HRP-HBeAb monoclonal antibodies, detect recombinant hepatitis b virus e antigens
Main calibration object CAL1-CAL7.After 37 degree of calculating water-bath 7 days, 14 days, antibody magnetic particle signal retention rate.
The main calibration object preparation method of recombinant hepatitis b virus e antigens is as follows：
CAL1:Sample dilution 4
CAL2:Sample dilution 4 is by 30000 times of recombinant hepatitis b virus e antigen diluents
CAL3:Sample dilution 4 is by 8000 times of recombinant hepatitis b virus e antigen diluents
CAL4:Sample dilution 4 is by 1250 times of recombinant hepatitis b virus e antigen diluents
CAL5:Sample dilution 4 is by 225 times of recombinant hepatitis b virus e antigen diluents
CAL6:Sample dilution 4 is by 90 times of recombinant hepatitis b virus e antigen diluents
CAL7:Sample dilution 4 is by 20 times of recombinant hepatitis b virus e antigen diluents
Antibody coating magnetic particle preparation method is as follows：
One-step method is coated with the anti-magnetic particle of HBeAg monoclonal antibodies -1：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 20mg/ml to make its concentration；
2nd, activation coating：EDC.HCL solution is prepared in enchashment, by EDC.HCL and magnetic particle mass ratio 1:5 ratio is to resuspension
EDC.HCL solution is added in good magnetic particle, is vortexed and mixes；It is again 1 by antibody and magnetic particle mass ratio:50 coating ratio to
Being added in the good magnetic particle of above-mentioned activation needs the coated anti-antibody of HBeAg monoclonal antibodies -1, is vortexed after mixing, and room temperature is horizontal mixed
Even 12 hours；
3rd, close：1mol/L Tris solution, 10%BSA solution mixing confining liquids, room temperature water are added into above-mentioned magnetic particle
It is flat to mix 3 hours；
4th, clean：Above-mentioned magnetic particle is cleaned with the PBS solution containing Tween 5 times, completes anti-HBeAg monoclonal antibodies -1
Antibody is coated with magnetic particle.
Two-step method is coated with the anti-magnetic particle of HBeAg monoclonal antibodies -1：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 20mg/ml to make its concentration；
2nd, activate：EDC.HCL solution is matched somebody with somebody in enchashment, by EDC.HCL and magnetic particle mass ratio 1:5 ratio is to the magnetic being resuspended
EDC.HCL solution is added in particulate, is vortexed after mixing, room temperature is horizontal to be mixed 30 minutes；
3rd, it is coated with：It is 1 by antibody and magnetic particle mass ratio:50 coating ratio adds into the good magnetic particle of above-mentioned activation
The coated anti-antibody of HBeAg monoclonal antibodies -1 is needed, is vortexed after mixing, room temperature is horizontal to be mixed 12 hours；
4th, close：1mol/L Tris solution, 10%BSA solution mixing confining liquids, room temperature water are added into above-mentioned magnetic particle
It is flat to mix 3 hours；
5th, clean：Above-mentioned magnetic particle is cleaned with the PBS solution containing Tween 5 times, completes anti-HBeAg monoclonal antibodies -1
Antibody is coated with magnetic particle.
The inventive method is coated with the anti-magnetic particle of HBeAg monoclonal antibodies -1：
1st, clean：Appropriate magnetic particle is taken, magnetic particle is cleaned 4 times with 0.1mol/L MES, then is resuspended with 0.1mol/L MES
Magnetic particle, it is 20mg/ml to make its concentration；
2nd, activate：EDC.HCL solution is matched somebody with somebody in enchashment, by EDC.HCL and magnetic particle mass ratio 1:5 ratio is to the magnetic being resuspended
EDC.HCL solution is added in particulate, is vortexed after mixing, room temperature is horizontal to be mixed 30 minutes；
3rd, it is coated with：It is 1 by antibody and magnetic particle mass ratio:50 coating ratio adds into the good magnetic particle of above-mentioned activation
The coated anti-antibody of HBeAg monoclonal antibodies -1 is needed, is vortexed after mixing, room temperature is horizontal to be mixed 12 hours；
4th, it is re-activated：Fresh EDC.HCL solution is taken, by EDC.HCL and magnetic particle mass ratio 1:5
Ratio adds EDC.HCL solution again into the magnetic particle of the good anti-antibody of HBeAg monoclonal antibodies -1 of coating,
It is vortexed after mixing, room temperature is horizontal to be mixed 2 hours；
5th, close：1mol/L Tris solution, the mixing closing of 10%BSA solution are added into above-mentioned magnetic particle
Liquid, room temperature is horizontal to be mixed 3 hours；
6th, clean：Above-mentioned magnetic particle is cleaned with the PBS solution containing Tween 5 times, and it is mono- to complete anti-HBeAg
The antibody of clonal antibody -1 is coated with magnetic particle.
Using the anti-antibody magnetic particle of HBeAg monoclonal antibodies -1 of above-mentioned three kinds coating technique, detection restructuring second
Signal retention rate is shown in Table 3 after Hepatitis virus e antigen signals value and 37 DEG C of water-baths.
Table 3
Result above shows, the anti-magnetic particle detected signal value of HBeAg monoclonal antibodies -1 is coated with compared with two steps using one-step method
Method and the inventive method are low, therefore do not carry out 37 DEG C of water-baths to one-step method coating magnetic particle.And by anti-HBeAg monoclonal antibodies -1
The antigen magnetic of the higher two-step method of magnetic particle detected signal value and the inventive method is micro-
After grain is positioned over 37 DEG C of water-baths 7 days, 14 days, the coated anti-magnetic particle of HBeAg monoclonal antibodies -1 of the inventive method
Signal retention rate is compared with two-step method height.
Embodiment 4
Using the inventive method by RNP antigen coat magnetic particles, during coating, activating solution described in step 2 and magnetic
Particle mass ratio is 1:21；Antigen described in step 3 or antibody and magnetic particle mass ratio are 1:2200；It is living described in step 4
It is 1 to change liquid with magnetic particle mass ratio:21.Supporting HRP- anti-human IgG antibodies again, detect the main calibration object of RNP projects.
RNP magnetic particles are coated with using above-mentioned coating ratio, supporting HRP- anti-human IgG antibodies, detect the main calibration of RNP projects
Product signal value is shown in Table 4：
Table 4
| Main calibration object | The main calibration object signals of RNP |
| CAL1 | 210 |
| CAL2 | 8913 |
| CAL3 | 67584 |
| CAL4 | 217485 |
| CAL5 | 484758 |
| CAL6 | 675886 |
The signal value that above scheme detects is far below in embodiment 1, using the letter of the inventive method testing calibration product
Number value.
Embodiment 5
Using the inventive method by SCL-70 antigen coat magnetic particles, during coating, activating solution described in step 2
It is 6 with magnetic particle mass ratio:1；Antigen described in step 3 or antibody and magnetic particle mass ratio are 1:48；It is living described in step 4
It is 6 to change liquid with magnetic particle mass ratio:1.Supporting HRP- anti-human IgG antibodies again, detect the main calibration object of SCL-70 projects.
SCL-70 magnetic particles are coated with using above-mentioned coating ratio, supporting HRP- anti-human IgG antibodies, detect SCL-70 projects
Main calibration object signal value is shown in Table 5：
Table 5
| Main calibration object | The main calibration object signals of SCL-70 |
| CAL1 | 117 |
| CAL2 | 6775 |
| CAL3 | 57859 |
| CAL4 | 94754 |
| CAL5 | 191457 |
| CAL6 | 347584 |
The signal value that above scheme detects is far below in example 2, using the letter of the inventive method testing calibration product
Number value.
Claims (15)
1. the method for antigen or antibody coating magnetic particle, it is characterised in that the described method comprises the following steps：
Step 1：Magnetic particle is taken, magnetic particle is resuspended with buffer A；
Step 2：Activating solution is taken, is added in the magnetic particle solution that step 1 has been resuspended, is mixed；
Step 3：Antigen or antibody are added in the magnetic particle solution that step 2 has activated, mixed；
Step 4：Activating solution is taken, is added in step 3 solution, is mixed；
Step 5：Confining liquid is taken, is added in step 4 solution, is mixed；
Step 6：Buffer B is taken, the magnetic particle of cleaning step five, completes antigen or antibody coating magnetic particle.
2. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that described Step 2: living in four
Change liquid is EDC.HCL or DCC solution.
3. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that buffer solution in the step 1
A is MES solution.
4. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that confining liquid in the step 5
For Tris or BSA or the one or more of casein solution.
5. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that buffer solution in the step 6
B is TBST or PBS or the PBS solution containing Tween.
6. the method for antigen or antibody coating magnetic particle as described in any one of claim 1 to 5, it is characterised in that the magnetic is micro-
Grain average grain diameter is 1 μm~3 μm.
7. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that buffer solution in the step 1
Magnetic particle concentration after A resuspension magnetic particles is 2mg/ml~20mg/ml.
8. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that activating solution in the step 2
Middle EDC.HCL or DCC are 1 with magnetic particle mass ratio:20~5:1.
9. antigen as claimed in claim 1 or antibody coating magnetic particle method, it is characterised in that in the step 3 antigen or
Antibody is 1 with magnetic particle mass ratio:2000~1:50.
10. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that activated in the step 4
EDC.HCL or DCC and magnetic particle mass ratio are 1 in liquid:20~5:1.
11. the method for antigen as claimed in claim 1 or antibody coating magnetic particle, it is characterised in that the magnetic in the step 1
Particulate had been cleaned before being resuspended by buffer A by buffer A.
A kind of 12. kit, it is characterised in that the kit includes antigen or the coated magnetic particle of antibody, the antigen or
The method that the coated magnetic particle of antibody is coated with magnetic particle by any one of the claim 1-11 antigens or antibody is prepared.
13. magnetic particle prepared by the method for any one of the claim 1-11 antigens or antibody coating magnetic particle is in chemiluminescence
Application in immunoassay.
14. application as claimed in claim 13, it is characterised in that the magnetic particle is used for various specific totals in human serum and resisted
Body, specific IgG, IgM antibody-likes, the chemiluminescence immune assay of specific antigen.
15. application as claimed in claim 13, it is characterised in that the magnetic particle be used for RNP antigens, SCL-70 antigens,
The chemistry hair of at least one of JO-1 antigens, HIV antigens, HCV antigens, HBeAg antibody, insulin, FT3, FT4 and Renin
Light immunoassay.
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| CN201710507484.2A CN107462708B (en) | 2017-06-28 | 2017-06-28 | Method, application and the kit of antigen or antibody coating magnetic particle |
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| CN107462708B CN107462708B (en) | 2018-06-01 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113687063A (en) * | 2021-07-29 | 2021-11-23 | 南昌大学 | Glycoprotein dynamic light scattering immunization method based on phenylboronic acid crosslinking agent |
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