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CN113917133A - Method for labeling antibody by microspheres - Google Patents

Method for labeling antibody by microspheres Download PDF

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CN113917133A
CN113917133A CN202111164677.5A CN202111164677A CN113917133A CN 113917133 A CN113917133 A CN 113917133A CN 202111164677 A CN202111164677 A CN 202111164677A CN 113917133 A CN113917133 A CN 113917133A
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microspheres
solution
supernatant
centrifuge
vortex
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宋路红
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Multi Sciences Lianke Biotechnology Corporate Ltd
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Multi Sciences Lianke Biotechnology Corporate Ltd
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    • 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
    • G01N33/532—Production of labelled immunochemicals

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Abstract

The invention discloses a method for labeling an antibody by microspheres. In the invention, a vortex mixer is used for fully oscillating and dispersing the microspheres, and then the microspheres are counted by a flow method. Taking a proper amount of microspheres, adding 50mM MES (pH5pH6.0) for heavy suspension and washing, then adding the microspheres into 50mM MES (pH6.0) solution containing 5mg/ml EDC and 5mg/ml NHS for precipitation, carrying out vortex oscillation treatment on the microspheres by using a vortex oscillation machine, adding the microspheres into a centrifuge after the ultrasonic oscillation treatment is finished by using an ultrasonic oscillation machine, carrying out centrifugal treatment on the microspheres, taking out supernatant in a centrifugal liquid after the centrifugal process is finished, and abandoning the supernatant; then, washing the solution again by using clear water for one time for standby and activating; after washing with a 100mM MES (pH6.0) solution, the antibody was coupled to the microspheres by adding a 100mM MES (pH6.0) solution and an appropriate amount of the capture antibody; adding 250-300 mul of blocking solution for re-suspending and blocking, wherein the blocking solution is 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 PBS solution, so that the vacant sites on the surface of the microspheres are filled with BSA, and the detection antibody is not nonspecifically adsorbed to the microspheres and only combined with specific target protein, thereby improving the detection accuracy.

Description

Method for labeling antibody by microspheres
Technical Field
The invention belongs to the technical field of antibody labeling, and particularly relates to a method for labeling an antibody by microspheres.
Background
The antibody is labeled by enzyme, ferritin or colloidal gold, and is a method for preparing an immunoelectron microscope sample for observing the antigen-antibody immune complex. The immunolabeling technology is that some substances which are easy to be measured and have high sensitivity are labeled on specific antigen or antibody molecules, and the nature and content of the antigen or antibody in the reaction system can be displayed by the enhanced amplification effect of the labels. Antibody labeling is mainly used for antigen localization analysis, and in some cases, the antigen in a sample mixed with a large number of other molecules can be quantitatively detected. Because the antibody has high affinity with the corresponding antigen, the antibody with the easily-recognized marker can be used for positioning analysis of the antigen, and is an ideal rapid and cheap quantitative determination method.
However, in the conventional process of labeling antibodies, there are gaps between sample wells, and these gaps are not filled with the antibodies, and the antibodies are adsorbed in the empty wells, so that there are many non-specific signals, thereby affecting the accuracy of the experiment.
Disclosure of Invention
The invention aims to: in order to solve the above-mentioned problems, a method of labeling an antibody with a microsphere is provided.
The technical scheme adopted by the invention is as follows: the method for labeling the antibody by the microspheres is characterized by comprising the following steps: the method for labeling the antibody by the microspheres comprises the following steps:
s1, taking a centrifuge and 50 mul of microspheres, adding the microspheres into the centrifuge, beginning to perform centrifugal operation on the microspheres, and taking out supernatant in the centrifugate after the centrifugal process is finished, and discarding the supernatant;
s2 taking vortex oscillator, ultrasonic oscillator, adding 100 μ l NaH2PO4Carrying out vortex oscillation treatment on the microsphere solution by using a vortex oscillation machine, then carrying out ultrasonic oscillation on the microsphere solution by using an ultrasonic oscillation machine, adding the microsphere solution into a centrifugal machine after the treatment is finished, carrying out centrifugal treatment on the microsphere solution, and taking out supernatant in centrifugal liquid after the centrifugal process is finished, wherein the supernatant is not used for abandoning;
s3, adding 80 ul of 50MES buffer solution into the solution obtained in the step S2, carrying out vortex oscillation treatment by using a vortex oscillation machine, carrying out ultrasonic oscillation on the solution by using an ultrasonic oscillation machine, adding 10 ul of 50mg/ml EDC solution and 10 ul of 50mg/ml ulfo-NHS solution after the treatment is finished, and carrying out vortex oscillation and uniform mixing;
s4, after the step S3 is finished, oscillating and incubating for 20 minutes in a dark place at room temperature, taking the centrifuge after the incubation is finished, centrifuging for 10 minutes at the centrifugation speed controlled to be 1000Xg, and after the centrifugation process is finished, taking out the supernatant in the centrifugate and discarding the supernatant;
s5, adding 50mM MES (pH5.0) for heavy suspension and precipitation, carrying out vortex oscillation treatment on the precipitate by using a vortex oscillation machine, adding the microsphere solution into a centrifuge after the ultrasonic oscillation treatment is finished by using the ultrasonic oscillation machine, carrying out centrifugal treatment on the precipitate, taking out supernatant in the centrifugate after the centrifugal process is finished, and discarding the supernatant; then, repeatedly washing the solution once again by using clear water for later use;
s6, adding 70 mul of anti-human IL-6 antibody with the concentration of 500 mug/ml into a desalting column for desalting once;
s7, adding the desalted IL-6 antibody into the microspheres, carrying out vortex oscillation treatment on the microspheres by using a vortex oscillator, and after the microspheres are uniformly mixed, carrying out oscillation incubation for 2 hours at room temperature in a dark place
S8, after finishing incubation for 2 hours, adding the microsphere solution into a centrifuge for centrifugation, and after the centrifugation process is finished, taking out the supernatant in the centrifugate and discarding the supernatant;
s9, adding confining liquid, carrying out vortex oscillation treatment on the confining liquid by using a vortex oscillation machine, then adding the microsphere solution into a centrifuge after the ultrasonic oscillation treatment of the confining liquid by using the ultrasonic oscillation machine is finished, carrying out centrifugal treatment on the confining liquid, and taking out supernatant liquid in the centrifugate after the centrifugal process is finished, and abandoning the supernatant liquid;
s10, centrifuging and washing twice by using confining liquid for standby;
and S11, adding 250-300 mul of confining liquid for resuspension, counting on a flow machine, bottling and storing the mixed liquid, and finally finishing the whole process of marking the antibody.
In a preferred embodiment, in the step S1, the content of the microspheres in the selected 50 μ l microspheres is 4 × 106The speed of centrifugation was controlled to 1000Xg, and the time of centrifugation was controlled to 10 minutes.
In a preferred embodiment of the present invention,in the step S2, NaH is added after vortex oscillation2PO4In the process of (3), NaH is controlled2PO4The pH of the solution was 6.2, the time for vortex oscillation was 20 seconds, the time for sonication was 20 seconds, and the speed of centrifugation was controlled at 1000 Xg.
In a preferred embodiment, in step S3, the time for vortex oscillation and sonication is 20 seconds for 80. mu.l of 50MES buffer and for 10. mu.l of 50mg/ml EDC solution and 10. mu.l of 50mg/ml ulfo-NHS solution.
In a preferred embodiment, in step S5, the time of rotational oscillation is 30 seconds, the time of ultrasonic treatment is 30 seconds, and the speed of centrifugation is controlled to be 1000 xg.
In a preferred embodiment, in step S8, the centrifugation speed is controlled to be 1000xg, and the centrifugation time is 10 minutes.
In a preferred embodiment, in the step S9, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution.
In a preferred embodiment, in the step S10, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution, and the rate of centrifugal washing is controlled to be 1000 Xg.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. in the invention, the confining liquid is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution, and can effectively react with the surface of the microsphere, so that cavities can be filled with BSA or casein, and thus, antibody protein can not be nonspecifically adsorbed on the membrane but only can be combined with specific protein, thereby improving the marking accuracy and the reaction rate in the subsequent use process.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows:
a method of microsphere labeling an antibody, the method comprising the steps of:
s1, taking a centrifuge and 50 mul of microspheres, adding the microspheres into the centrifuge, beginning to perform centrifugal operation on the microspheres, and taking out supernatant in the centrifugate after the centrifugal process is finished, and discarding the supernatant; in S1, the content of the selected microspheres in 50 mul microspheres is 4 multiplied by 106Controlling the speed of centrifugation to be 1000Xg and the time of centrifugation to be 10 minutes;
s2 taking vortex oscillator, ultrasonic oscillator, adding 100 μ l NaH2PO4Carrying out vortex oscillation treatment on the microsphere solution by using a vortex oscillation machine, then carrying out ultrasonic oscillation on the microsphere solution by using an ultrasonic oscillation machine, adding the microsphere solution into a centrifugal machine after the treatment is finished, carrying out centrifugal treatment on the microsphere solution, and taking out supernatant in centrifugal liquid after the centrifugal process is finished, wherein the supernatant is not used for abandoning; in step S2, after vortexing, NaH was added2PO4In the process of (3), NaH is controlled2PO4The pH of the solution is 6.2, the vortex oscillation time is 20 seconds, the ultrasonic treatment time is 20 seconds, and the centrifugation speed is controlled to be 1000 Xg;
s3, adding 80 ul of 50MES buffer solution into the solution obtained in the step S2, carrying out vortex oscillation treatment by using a vortex oscillation machine, carrying out ultrasonic oscillation on the solution by using an ultrasonic oscillation machine, adding 10 ul of 50mg/ml EDC solution and 10 ul of 50mg/ml ulfo-NHS solution after the treatment is finished, and carrying out vortex oscillation and uniform mixing; in step S3, the vortex oscillation time was 20 seconds and the sonication time was 20 seconds after adding 80. mu.l of 50MES buffer and after adding 10. mu.l of 50mg/ml EDC solution and 10. mu.l of 50mg/ml ulfo-NHS solution;
s4, after the step S3 is finished, oscillating and incubating for 20 minutes in a dark place at room temperature, taking the centrifuge after the incubation is finished, centrifuging for 10 minutes at the centrifugation speed controlled to be 1000Xg, and after the centrifugation process is finished, taking out the supernatant in the centrifugate and discarding the supernatant;
s5, adding 50mM MES (pH5.0) for heavy suspension and precipitation, carrying out vortex oscillation treatment on the precipitate by using a vortex oscillation machine, adding the microsphere solution into a centrifuge after the ultrasonic oscillation treatment is finished by using the ultrasonic oscillation machine, carrying out centrifugal treatment on the precipitate, taking out supernatant in the centrifugate after the centrifugal process is finished, and discarding the supernatant; then, repeatedly washing the solution once again by using clear water for later use; in step S5, the time of rotational oscillation is 30 seconds, the time of ultrasonic treatment is 30 seconds, and the speed of centrifugation is controlled to 1000 xg;
s6, adding 70 mul of anti-human IL-6 antibody with the concentration of 500 mug/ml into a desalting column for desalting once;
s7, adding the desalted IL-6 antibody into the microspheres, carrying out vortex oscillation treatment on the microspheres by using a vortex oscillator, and after the microspheres are uniformly mixed, carrying out oscillation incubation for 2 hours at room temperature in a dark place
S8, after finishing incubation for 2 hours, adding the microsphere solution into a centrifuge for centrifugation, and after the centrifugation process is finished, taking out the supernatant in the centrifugate and discarding the supernatant; in step S8, the speed of centrifugation is controlled to 1000xg, and the time of centrifugation is 10 minutes;
s9, adding confining liquid, carrying out vortex oscillation treatment on the confining liquid by using a vortex oscillation machine, then adding the microsphere solution into a centrifuge after the ultrasonic oscillation treatment of the confining liquid by using the ultrasonic oscillation machine is finished, carrying out centrifugal treatment on the confining liquid, and taking out supernatant liquid in the centrifugate after the centrifugal process is finished, and abandoning the supernatant liquid; in step S9, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution, and 0.02% Tween20 solution;
s10, centrifuging and washing twice by using confining liquid for standby; in step S10, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution, and the rate of centrifugal washing is controlled to be 1000 Xg;
s11, adding 250 mul of confining liquid for re-suspension, counting on a flow machine, bottling and storing the mixed liquid, and finally finishing the whole process of marking the antibody; the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution, and can effectively react with the surface of the microsphere, so that the cavities can be filled with BSA or casein, and thus, the antibody protein can not be nonspecifically adsorbed on the membrane but only can be combined with specific protein, thereby improving the marking accuracy and also improving the reaction rate in the subsequent use process.
Example two:
a method of microsphere labeling an antibody, the method comprising the steps of:
s1, taking a centrifuge and 50 mul of microspheres, adding the microspheres into the centrifuge, beginning to perform centrifugal operation on the microspheres, and taking out supernatant in the centrifugate after the centrifugal process is finished, and discarding the supernatant; in S1, the content of the selected microspheres in 50 mul microspheres is 4 multiplied by 106Controlling the speed of centrifugation to be 1000Xg and the time of centrifugation to be 10 minutes;
s2 taking vortex oscillator, ultrasonic oscillator, adding 100 μ l NaH2PO4Carrying out vortex oscillation treatment on the microsphere solution by using a vortex oscillation machine, then carrying out ultrasonic oscillation on the microsphere solution by using an ultrasonic oscillation machine, adding the microsphere solution into a centrifugal machine after the treatment is finished, carrying out centrifugal treatment on the microsphere solution, and taking out supernatant in centrifugal liquid after the centrifugal process is finished, wherein the supernatant is not used for abandoning; in step S2, after vortexing, NaH was added2PO4In the process of (3), NaH is controlled2PO4The pH of the solution is 6.2, the vortex oscillation time is 20 seconds, the ultrasonic treatment time is 20 seconds, and the centrifugation speed is controlled to be 1000 Xg;
s3, adding 80 ul of 50MES buffer solution into the solution obtained in the step S2, carrying out vortex oscillation treatment by using a vortex oscillation machine, carrying out ultrasonic oscillation on the solution by using an ultrasonic oscillation machine, adding 10 ul of 50mg/ml EDC solution and 10 ul of 50mg/ml ulfo-NHS solution after the treatment is finished, and carrying out vortex oscillation and uniform mixing; in step S3, the vortex oscillation time was 20 seconds and the sonication time was 20 seconds after adding 80. mu.l of 50MES buffer and after adding 10. mu.l of 50mg/ml EDC solution and 10. mu.l of 50mg/ml ulfo-NHS solution;
s4, after the step S3 is finished, oscillating and incubating for 20 minutes in a dark place at room temperature, taking the centrifuge after the incubation is finished, centrifuging for 10 minutes at the centrifugation speed controlled to be 1000Xg, and after the centrifugation process is finished, taking out the supernatant in the centrifugate and discarding the supernatant;
s5, adding 50mM MES (pH5.0) for heavy suspension and precipitation, carrying out vortex oscillation treatment on the precipitate by using a vortex oscillation machine, adding the microsphere solution into a centrifuge after the ultrasonic oscillation treatment is finished by using the ultrasonic oscillation machine, carrying out centrifugal treatment on the precipitate, taking out supernatant in the centrifugate after the centrifugal process is finished, and discarding the supernatant; then, repeatedly washing the solution once again by using clear water for later use; in step S5, the time of rotational oscillation is 30 seconds, the time of ultrasonic treatment is 30 seconds, and the speed of centrifugation is controlled to 1000 xg;
s6, adding 70 mul of anti-human IL-6 antibody with the concentration of 500 mug/ml into a desalting column for desalting once;
s7, adding the desalted IL-6 antibody into the microspheres, carrying out vortex oscillation treatment on the microspheres by using a vortex oscillator, and after the microspheres are uniformly mixed, carrying out oscillation incubation for 2 hours at room temperature in a dark place
S8, after finishing incubation for 2 hours, adding the microsphere solution into a centrifuge for centrifugation, and after the centrifugation process is finished, taking out the supernatant in the centrifugate and discarding the supernatant; in step S8, the speed of centrifugation is controlled to 1000xg, and the time of centrifugation is 10 minutes;
s9, adding confining liquid, carrying out vortex oscillation treatment on the confining liquid by using a vortex oscillation machine, then adding the microsphere solution into a centrifuge after the ultrasonic oscillation treatment of the confining liquid by using the ultrasonic oscillation machine is finished, carrying out centrifugal treatment on the confining liquid, and taking out supernatant liquid in the centrifugate after the centrifugal process is finished, and abandoning the supernatant liquid; in step S9, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution, and 0.02% Tween20 solution;
s10, centrifuging and washing twice by using confining liquid for standby; in step S10, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution, and the rate of centrifugal washing is controlled to be 1000 Xg;
s11, adding 250 mul of confining liquid for re-suspension, counting on a flow machine, bottling and storing the mixed liquid, and finally finishing the whole process of marking the antibody; the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution, and can effectively react with the surface of the microsphere, so that the cavities can be filled with BSA or casein, and thus, the antibody protein can not be nonspecifically adsorbed on the membrane but only can be combined with specific protein, thereby improving the marking accuracy and also improving the reaction rate in the subsequent use process.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (8)

1.微球标记抗体的方法，其特征在于：所述微球标记抗体的方法包括以下步骤：1. a method for labeling antibodies with microspheres, characterized in that: the method for labeling antibodies with microspheres comprises the following steps: S1:将微球溶液用涡旋混合仪充分振荡，使之分散成单个微球，取适量微球溶液，稀释后于流式细胞仪上进行计数。S1: Fully shake the microsphere solution with a vortex mixer to disperse it into a single microsphere, take an appropriate amount of the microsphere solution, dilute it and count it on a flow cytometer. 取来离心机和50μl微球，将微球加入到离心机的内部，开始对微球进行离心操作，离心过程结束之后，取出离心液中的上清液，将其废弃不用；Take the centrifuge and 50 μl of microspheres, add the microspheres to the inside of the centrifuge, and start centrifuging the microspheres. After the centrifugation process is over, take out the supernatant in the centrifuge and discard it; S2:取适量微球(如1×106个)来涡旋振荡机，超声震荡机，加入100μlNaH2PO4，使用涡旋振荡机对其进行涡旋振荡处理，之后使用超声震荡机对其进行超声震荡，处理结束之后，将微球溶液加入到离心机的内部，对其进行1500×g离心5分钟处理，弃上清，加入100μl 50mMMES(pH6.0)缓冲液，涡旋振荡，1500×g离心5分钟，弃上清离心过程结束之后，取出离心液中的上清液，将其废弃不用；S2: Take an appropriate amount of microspheres (such as 1×106) to a vortex shaker, an ultrasonic shaker, add 100 μl NaH 2 PO 4 , use a vortex shaker to vortex them, and then use an ultrasonic shaker to vortex them. Ultrasonic vibration, after the treatment, add the microsphere solution to the inside of the centrifuge, centrifuge it at 1500×g for 5 minutes, discard the supernatant, add 100 μl 50mMMES (pH6.0) buffer, vortex, 1500× Centrifuge at g for 5 minutes, discard the supernatant. After the centrifugation process is over, take out the supernatant in the centrifuge and discard it; S3:取来步骤S2中的沉淀，加入100μl含5mg/ml EDC和5mg/ml sulfo-NHS的50mM MES(pH6.0)缓冲液，涡旋振荡，室温避光孵育20分钟；S3: take the precipitate in step S2, add 100 μl of 50 mM MES (pH 6.0) buffer containing 5 mg/ml EDC and 5 mg/ml sulfo-NHS, vortex, and incubate at room temperature for 20 minutes in the dark; S4:步骤S3结束之后，1500×g离心5分钟，弃上清。加入200μl 100mM MES(pH6.0)缓冲液，涡旋振荡，1500×g离心5分钟，弃上清；S4: After the end of step S3, centrifuge at 1500×g for 5 minutes, and discard the supernatant. Add 200 μl of 100 mM MES (pH 6.0) buffer, vortex, centrifuge at 1500 × g for 5 minutes, and discard the supernatant; S5:加入300μl 100mM MES(pH6.0)缓冲液，涡旋振荡；S5: add 300 μl of 100 mM MES (pH 6.0) buffer, vortex; S6:S6: S7:取5μg抗人IL-6捕获抗体加入微球中，室温避光振荡孵育2小时；S7: Add 5 μg of anti-human IL-6 capture antibody to the microspheres, and incubate for 2 hours at room temperature with shaking in the dark; S8:孵育结束后，将微球溶液于1500×g离心5分钟，弃上清。；S8: After the incubation, the microsphere solution was centrifuged at 1500×g for 5 minutes, and the supernatant was discarded. ; S9:加入200μl封闭液，室温避光振荡孵育2小时，孵育结束后，于1500×g离心5分钟，弃上清。；S9: Add 200 μl of blocking solution, and incubate at room temperature for 2 hours with shaking in the dark. After the incubation, centrifuge at 1500×g for 5 minutes, and discard the supernatant. ; S10:加入200μl封闭液，涡旋振荡，于1500×g离心5分钟，弃上清；S10: Add 200 μl blocking solution, vortex, centrifuge at 1500×g for 5 minutes, discard the supernatant; S11:加入200μl封闭液重悬，完成整个标记抗体的过程。S11: Add 200 μl of blocking solution to resuspend to complete the whole process of labeling antibodies. 2.如权利要求1所述的微球标记抗体的方法，其特征在于：所述步骤S1中，选用的50μl微球内部微球的含量为4×106个，离心的速度控制为1000xg，离心的时间控制为10分钟。2. The method for labeling antibodies with microspheres as claimed in claim 1, wherein in the step S1, the content of the microspheres in the selected 50 μl microspheres is 4× 10 , and the centrifugal speed is controlled to be 1000×g, The time of centrifugation was controlled to 10 minutes. 3.如权利要求1所述的微球标记抗体的方法，其特征在于：所述步骤S2中，涡旋振荡后，加入NaH2PO4的过程中，控制NaH2PO4溶液的pH在6.2，涡旋振荡的时间为20秒，超声处理的时间为20秒，离心的速度控制为1000xg。3. The method for labeling antibodies by microspheres as claimed in claim 1, wherein in the step S2, after vortexing, in the process of adding NaH 2 PO 4 , the pH of the NaH 2 PO 4 solution is controlled at 6.2 , the time of vortexing was 20 seconds, the time of sonication was 20 seconds, and the speed of centrifugation was controlled to 1000×g. 4.如权利要求1所述的微球标记抗体的方法，其特征在于：所述步骤S3中，加入80μl50MES缓冲液之后和加入10μl50mg/mlEDC溶液和10μl50mg/mlsulfo-NHS溶液之后的涡旋振荡的时间都为20秒，超声处理的时间都为20秒。4. The method for labeling antibodies by microspheres as claimed in claim 1, wherein in the step S3, after adding 80 μl 50MES buffer and adding 10 μl 50mg/ml EDC solution and 10 μl 50mg/ml sulfo-NHS solution The times were all 20 seconds and the sonication time was all 20 seconds. 5.如权利要求1所述的微球标记抗体的方法，其特征在于：所述步骤S5中，旋振荡的时间为30秒，超声处理的时间为30秒，离心的速度控制为1000xg。5. The method for labeling antibodies by microspheres as claimed in claim 1, wherein in the step S5, the time of spin oscillation is 30 seconds, the time of ultrasonic treatment is 30 seconds, and the speed of centrifugation is controlled to be 1000×g. 6.如权利要求1所述的微球标记抗体的方法，其特征在于：所述步骤S8中，离心的速度控制为1000xg，离心处理的时间为10分钟。6 . The method for labeling antibodies by microspheres according to claim 1 , wherein in the step S8 , the speed of centrifugation is controlled to be 1000×g, and the time of centrifugation is 10 minutes. 7 . 7.如权利要求1所述的微球标记抗体的方法，其特征在于：所述步骤S9中，封闭液为99.88％PBS溶液、0.1％BSA溶液和0.02％Tween20溶液的混合物。7. The method for labeling antibodies by microspheres according to claim 1, wherein in the step S9, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution. 8.如权利要求1所述的微球标记抗体的方法，其特征在于：所述步骤S10中，封闭液为99.88％PBS溶液、0.1％BSA溶液和0.02％Tween20溶液的混合物，离心洗涤的速率为控制为1000xg。8. The method for labeling antibodies by microspheres as claimed in claim 1, wherein in the step S10, the blocking solution is a mixture of 99.88% PBS solution, 0.1% BSA solution and 0.02% Tween20 solution, and the speed of centrifugal washing is For the control is 1000xg.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101144815A (en) * 2006-09-13 2008-03-19 广州市达瑞抗体工程技术有限公司 Preparation method of liquid phase protein chip
US20130295688A1 (en) * 2010-11-05 2013-11-07 Ryan C. Bailey Optical analyte detection systems and methods of use
CN109187956A (en) * 2018-09-05 2019-01-11 杭州莱和生物技术有限公司 A kind of antibody labeling method and its application of the coupling of time-resolved fluorescence microballoon

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101144815A (en) * 2006-09-13 2008-03-19 广州市达瑞抗体工程技术有限公司 Preparation method of liquid phase protein chip
US20130295688A1 (en) * 2010-11-05 2013-11-07 Ryan C. Bailey Optical analyte detection systems and methods of use
CN109187956A (en) * 2018-09-05 2019-01-11 杭州莱和生物技术有限公司 A kind of antibody labeling method and its application of the coupling of time-resolved fluorescence microballoon

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Application publication date: 20220111