CN111077304A - Coupling method of carboxyl latex microspheres and antibody - Google Patents
Coupling method of carboxyl latex microspheres and antibody Download PDFInfo
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- 238000010168 coupling process Methods 0.000 title claims abstract description 31
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- 239000007995 HEPES buffer Substances 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- -1 N-hydroxyethyl thiosuccinimide Chemical compound 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
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Classifications
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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
-
- 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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Abstract
The invention discloses a coupling method of carboxyl latex microspheres and an antibody, and relates to the technical field of medical detection. The method effectively improves the monodispersity of the carboxyl latex microspheres, the concentration of the activated intermediates of the latex microspheres and the coupling efficiency of the carboxyl latex microspheres and the antibodies through the processes of pretreating the carboxyl latex microspheres, activating the carboxyl latex microspheres to form activated intermediates, and finally coupling the activated intermediates with the antibodies, avoids the self-coagulation of the latex microspheres, and maintains the activated state of the latex microspheres so as to improve the effective binding rate of the microspheres and the antibodies.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a coupling method of carboxyl latex microspheres and an antibody.
Background
The immunochromatography technology has been developed from the 90 s in the 20 th century to the present, and has become a mature rapid detection technology commonly used in clinical medicine, wherein the more common form is an immunochromatography test strip technology. The test strip is generally assembled by a PVC back plate, absorbent paper, a solid-phase nitrocellulose membrane (NC membrane), a combination pad and a sample pad. The working principle of the method is generally to adopt a direct competition method, an indirect competition method or a double-antibody sandwich method, and the key technology is to utilize a labeling material to couple an antibody so as to enable the antibody to develop color and further recognize an antigen substance.
Common chromogenic labels are enzymes, colloidal gold, and latex microspheres. Although the technology of using the enzyme as a marking material is mature, the enzyme is greatly influenced by temperature, the stability is poor, and the effective period of the product is short; colloidal gold is the most used marking material in the current commercial test strip, but the signal method has high cost and cannot be used for accurate detection; the latex microspheres can form stable colloid in liquid, and compared with colloidal gold, the particles of the microspheres are larger, so that the using amount of antibodies can be saved, and the sensitivity is higher; meanwhile, large-scale production can be carried out, and the cost is lower; the stability and the sensitivity are better. Therefore, the covalent bonding between the latex microspheres with different groups on the surface and the antibody to form a complex with a stable structure is a hot spot of current research in the fields of disease diagnosis, biological medicine and the like.
However, the latex microspheres are prone to coagulation before activation, and hydrolysis is prone to occur when the pH value is increased after activation, so that the coupling efficiency of the latex microspheres and the antibody is low, and the application of the latex microsphere marker in clinical immunoassay is difficult to satisfy.
Disclosure of Invention
The technical problem to be solved by the invention is to solve the defects in the prior art, and provide a method for coupling carboxyl latex microspheres and antibodies, so that the coupling efficiency of the latex microspheres and the antibodies is improved.
In order to solve the above problems, the present invention proposes the following technical solutions:
a coupling method of carboxyl latex microspheres and antibodies comprises the following steps:
s1, fully mixing a stock solution of carboxyl latex microspheres with the mass fraction of 4% with an activation buffer solution according to the volume ratio of 1:15-25, centrifuging, discarding the supernatant, adding the activation buffer solution with the volume of 4 times that of the stock solution of the carboxyl latex microspheres into the precipitate, mixing again, performing ultrasonic treatment until the carboxyl latex microspheres in the solution are in a monodisperse state to obtain a microsphere suspension, and storing the microsphere suspension at 4 ℃ for later use;
s2, quickly and fully mixing 100g/L EDC aqueous solution, 100g/L Sulfo-NHS aqueous solution and the microsphere suspension according to the volume ratio of 0.8-1.2:5.5-6.5:65-75, and reacting for 30min to obtain a first mixed solution;
s3, performing ultrasonic treatment on the first mixed solution until the carboxyl latex microspheres in the first mixed solution are in a monodisperse state, performing centrifugal washing to obtain a precipitation solution, adding Tween-20 with the mass fraction of 10% into the precipitation solution, uniformly mixing to obtain an activated microsphere solution, and storing the activated microsphere solution at 4 ℃ for later use, wherein the mass fraction of the Tween-20 in the activated microsphere solution is 0.1-0.2%;
s4, mixing 0.1-1 ug/mu L of antibody solution with the activated microsphere solution according to the volume ratio of 3-4:1, and carrying out coupling reaction for more than 2h to obtain a microsphere-antibody marker solution;
s5, adding 1mol/L ethanolamine solution into the microsphere-antibody marker solution, and uniformly mixing in a rotating manner to obtain a third mixed solution, wherein the concentration of ethanolamine in the third mixed solution is 5-20 mmol/L;
s6, carrying out ultrasonic and centrifugal washing on the third mixed solution to obtain a microsphere-antibody marker precipitate, and adding a preservation solution into the microsphere-antibody marker precipitate for heavy suspension to obtain carboxyl latex microsphere-antibody marker heavy suspension containing 0.5% of mass fraction.
Further, step S1 is preceded by: and (3) carrying out ultrasonic treatment on the carboxyl latex microsphere liquid for 3-10 min.
Further, the centrifugal washing in the step S3 to obtain the precipitation solution specifically includes steps S301 to S302:
s301, performing first centrifugation on the second mixed solution after ultrasonic treatment at 8000-12000rpm for 10-20min, discarding the supernatant, and re-suspending the precipitate with 1mL of SDS solution to obtain a first re-suspension solution, wherein the mass fraction of the SDS solution is 0.005%;
s302, centrifuging the first resuspension solution for the second time at 8000-12000rpm for 10-20min, discarding the supernatant, adding purified water with the volume consistent with that of the stock solution of the carboxyl latex microspheres to carry out resuspension precipitation to obtain a second resuspension solution, and carrying out ultrasonic treatment on the second resuspension solution until the carboxyl latex microspheres in the second resuspension solution are in a monodisperse state to obtain the precipitation solution.
Further, the step S6 of centrifugal washing to obtain the microsphere-antibody label precipitate includes the steps S601-602:
s601, carrying out first centrifugation on the third mixed solution after the ultrasonic treatment at 8000-12000rpm for 10-20min, discarding the supernatant, and re-suspending the precipitate with 1mL of preservation solution to obtain a third suspension;
s602, carrying out second centrifugation on the third suspension at 8000-.
Further, after the step S6, the method further comprises the step of preserving the carboxyl latex microsphere-antibody marker resuspension at 4 ℃.
Further, the antibody solution is prepared by antibody stock solution and antibody buffer solution, wherein the antibody buffer solution is 100mmol/L HEPES buffer solution or 100mmol/L PB buffer solution.
Further, the pH of the activation buffer was 6, and 19 to 20g of MES and 0.001 to 1g of SDS were included in 1L of the activation buffer.
Further, the pH of the storage solution was 8.5, and the storage solution was prepared by adding 0.5g of BSA, 0.5g of Tween-20 and 0.05g of ProClin300 to 100mL of 10mmol/L Tris-HCl buffer.
Compared with the prior art, the invention can achieve the following technical effects:
(1) according to the coupling method of the carboxyl latex microspheres and the antibody, the surfactant SDS is added in the activation process to enhance the stability of the microspheres, and EDC and Sulfo-NHS are used for activation without adherence and coagulation, so that the monodispersity is ensured, and the ultrasonic treatment frequency is reduced; and the SDS can not react with EDC in the activation process of the latex microspheres, thus improving the activation efficiency of the microspheres; in the activation process, Sulfo-NHS is used for replacing the traditional NHS, so that the activated latex microspheres are still negatively charged and do not generate agglutination and precipitation;
(2) according to the coupling method of the carboxyl latex microspheres and the antibody, the pH of the suspension is not improved by washing the carboxyl latex microspheres with SDS (sodium dodecyl sulfate) solution after activation, so that the hydrolysis of an activated intermediate of the latex microspheres is greatly avoided; in the coupling reaction process, the Tween-20 serving as a mild surfactant can stabilize the activated latex microspheres, avoid coagulation caused by reduction of negative charges on the surfaces of the coupled latex microspheres, meanwhile, the low-concentration Tween-20 has no influence on the activity of antibodies, and the monodispersity of the latex microspheres is improved so as to improve the coupling efficiency.
(3) According to the coupling method of the carboxyl latex microspheres and the antibody, the buffer solution and 0.5% BSA in the preservation solution can protect the antibody coupled to the latex microspheres and prevent the antibody from denaturation, the latex is ensured to be in a monodisperse state by the low-concentration Tween-20, the trace amount of ProClin300 is used as a preservative, the latex microspheres marked with the antibody can be stably stored for more than 3 months at 4 ℃ without coagulation, and the activity of the antibody is hardly reduced.
(4) The coupling method of the carboxyl latex microspheres and the antibody is suitable for coupling the carboxyl latex microspheres and different antibodies, has wide application range, realizes qualitative and quantitative detection of antigen substances, and is easy to form large-scale preparation and production of test strips.
Detailed Description
The technical solutions in the examples will be clearly and completely described below. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. 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.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the embodiments of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the embodiments of the invention. As used in the description of embodiments of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The embodiment of the invention provides a coupling method of carboxyl latex microspheres and antibodies, which comprises the following steps:
s1, fully mixing 4% of carboxyl latex microsphere stock solution and an activation buffer solution according to the volume ratio of 1:15-25, centrifuging, discarding the supernatant, adding 4 times of the activation buffer solution of the carboxyl latex microsphere stock solution into the precipitate, mixing again, performing ultrasonic treatment until the carboxyl latex microspheres in the solution are in a monodisperse state to obtain microsphere suspension, and storing the microsphere suspension at 4 ℃ for later use.
The pH of the activation buffer was 6, and 19 to 20g of MES and 0.001 to 1g of SDS were contained in 1L of the activation buffer.
In step S1, the carboxyl latex microsphere solution is diluted and activated with an activation buffer solution to avoid self-coagulation of the carboxyl latex microspheres, and the carboxyl latex microspheres can be completely monodisperse by ultrasonic treatment.
For example, in one embodiment, the volume ratio of the carboxyl latex microsphere fluid to the activation buffer is 1: 20.
In one embodiment, the volume ratio of the carboxyl latex microsphere solution to the activation buffer solution is 1: 18.
In one embodiment, the volume ratio of the carboxyl latex microsphere solution to the activation buffer solution is 1: 23.
In another embodiment, in order to make the dispersion state of the carboxyl latex microspheres good and reduce the subsequent processing time, the carboxyl latex microsphere solution with the mass fraction of 4% can be subjected to ultrasonic processing for 3-10min in advance.
S2, quickly and fully mixing 100g/L EDC aqueous solution, 100g/L Sulfo-NHS aqueous solution and the microsphere suspension according to the volume ratio of 0.8-1.2:5.5-6.5:65-75, and reacting for 30min to obtain a first mixed solution;
in this embodiment, Sulfo-NHS is used to replace NHS in the conventional method, so that the activated carboxyl latex microspheres are still negatively charged and are not easily aggregated and precipitated. And SDS contained in the activation buffer solution does not react with EDC, so that the activation efficiency can be improved.
In one embodiment, the first mixture is prepared by rapidly mixing 100g/L EDC aqueous solution, 100g/L Sulfo-NHS aqueous solution and the microsphere suspension at a volume ratio of 1:5.8:68, and reacting for 30 min.
In one embodiment, the first mixture is prepared by rapidly mixing 100g/L EDC aqueous solution, 100g/L Sulfo-NHS aqueous solution and the microsphere suspension at a volume ratio of 1.1:6.1:72, and reacting for 30 min.
S3, performing ultrasonic treatment on the first mixed solution until the carboxyl latex microspheres in the first mixed solution are in a monodisperse state, performing centrifugal washing to obtain a precipitation solution, adding Tween-20 with the mass fraction of 10% into the precipitation solution, uniformly mixing to obtain an activated microsphere solution, and storing the activated microsphere solution at 4 ℃ for later use, wherein the mass fraction of the Tween-20 in the activated microsphere solution is 0.1-0.2%.
In the step, Tween-20 is added into the activated carboxyl latex microspheres, so that the activation state of the carboxyl latex microspheres can be stabilized, the monodispersity of the carboxyl latex microspheres is improved, and the coupling efficiency of subsequent reactions is improved.
In a specific embodiment, the centrifugation washing in step S3 to obtain the precipitation solution includes steps S301 to S302:
s301, performing first centrifugation on the second mixed solution after ultrasonic treatment at 8000-12000rpm for 10-20min, discarding the supernatant, and re-suspending the precipitate with 1mL of SDS solution to obtain a first re-suspension solution, wherein the mass fraction of the SDS solution is 0.005%;
s302, centrifuging the first resuspension solution for the second time at 8000-12000rpm for 10-20min, discarding the supernatant, adding purified water with the volume consistent with that of the stock solution of the carboxyl latex microspheres to carry out resuspension precipitation to obtain a second resuspension solution, and carrying out ultrasonic treatment on the second resuspension solution until the carboxyl latex microspheres in the second resuspension solution are in a monodisperse state to obtain the precipitation solution.
The activated carboxyl latex microspheres are washed and resuspended by SDS solution, so that the activated intermediate of the latex microspheres can not be hydrolyzed, and the stability of the carboxyl latex microspheres can be ensured.
S4, mixing 0.1-1 ug/mu L of antibody solution with the activated microsphere solution according to the volume ratio of 3-4:1, and carrying out coupling reaction for more than 2h to obtain a microsphere-antibody marker solution.
In one embodiment, the antibody solution is prepared from an antibody stock solution and an antibody buffer solution, wherein the antibody buffer solution is 100mmol/L HEPES buffer solution or 100mmol/L PB buffer solution.
The coupling reaction of the antibody and the carboxyl latex microspheres can be ensured to be more sufficient under the rotating condition. In one example, the coupling reaction was 2 h.
In one example, the coupling reaction was 2.5 h.
In one embodiment, the coupling reaction is for 4 h.
S5, adding 1mol/L ethanolamine solution into the microsphere-antibody marker solution, and uniformly mixing in a rotating manner to obtain a third mixed solution, wherein the concentration of ethanolamine in the third mixed solution is 5-20 mmol/L;
s6, carrying out ultrasonic and centrifugal washing on the third mixed solution to obtain a microsphere-antibody marker precipitate, and adding a preservation solution into the microsphere-antibody marker precipitate for heavy suspension to obtain carboxyl latex microsphere-antibody marker heavy suspension containing 0.5% of mass fraction.
The pH of the storage solution was 8.5, and the storage solution was prepared by adding 0.5g BSA, 0.5g Tween-20 and 0.05g ProClin300 to 100mL10mmol/L Tris-HCl buffer.
In other embodiments, the step S6 of centrifugal washing to obtain the microsphere-antibody label precipitate includes steps S601-602:
s601, carrying out first centrifugation on the third mixed solution after the ultrasonic treatment at 8000-12000rpm for 10-20min, discarding the supernatant, and re-suspending the precipitate with 1mL of preservation solution to obtain a third suspension;
s602, carrying out second centrifugation on the third suspension at 8000-.
In one embodiment, after the step S6, the method further comprises the step of storing the re-suspension of the carboxyl latex microsphere-antibody marker at 2-8 ℃.
It should be noted that the monodisperse state in the examples of the present invention means that the liquid containing the carboxyl group latex microspheres is observed under a 400x microscope without agglomeration and with large particles.
In the method for coupling the carboxyl latex microspheres and the antibody provided by the embodiment, through the processes of pretreating the carboxyl latex microspheres, activating the carboxyl latex microspheres to form an activated intermediate, and finally coupling the activated intermediate with the antibody, the monodispersity of the carboxyl latex microspheres, the concentration of the activated intermediate of the latex microspheres and the coupling efficiency of the carboxyl latex microspheres and the antibody are effectively improved, the latex microspheres are prevented from self-coagulation, and the activated state of the latex microspheres is maintained, so that the effective binding rate of the microspheres and the antibody is improved.
Another embodiment of the present invention provides a coupling method of carboxyl latex microspheres and antibodies, comprising the following steps:
1. pretreatment step of microspheres
1.1. Carrying out ultrasonic treatment on the carboxyl latex microspheres for 5 min;
1.2. taking 25uL of carboxyl latex microspheres (the mass fraction is 4 percent, namely 1mg), adding 475uL of activation buffer solution, fully and uniformly mixing, centrifuging at 10000rpm for 10min, and removing supernatant;
1.3. adding 100uL of activation buffer solution into the precipitate, uniformly blowing, sucking, and performing ultrasonic treatment until the microspheres are in a monodisperse state (no agglomeration and large particles are observed by a 400X microscope), wherein the microspheres are suspension for later use.
2. Microsphere activation step
2.1. Activation of
Quickly and uniformly mixing 1.4uL of 100g/L EDC solution and 8.6uL of 100g/L Sulfo-NHS solution, and quickly adding the mixture into 100uL microsphere suspension; quickly placing on a rotary mixer to rotate at 20rpm, mixing uniformly and reacting for 30 min.
2.2. Microscopic examination
The microspheres after activation should be free of agglomeration and large particles when observed under a 400x microscope.
2.3. Washing machine
Centrifuging at 10000rpm for 10min, discarding the supernatant, resuspending the precipitate with 1mL of 0.005% SDS solution, centrifuging at 10000rpm for 15min, and discarding the supernatant;
resuspending the precipitate in 25uL of purified water, adding 0.25uL of 10% Tween-20 solution to a final concentration of 0.1%, and performing ultrasonic treatment until the microspheres are in a monodisperse state (no agglomeration and large particles should exist after the microspheres are activated by observation under a 400X microscope); mixing uniformly for later use.
3. Antibody to latex microsphere coupling
Absorbing 60ug of antibody stock solution, adding 100mmol/L HEPES buffer solution or 100mmol/L PB buffer solution with a certain volume, diluting to 75uL, mixing well, adding into 2.3 microsphere suspension, and rapidly placing on a rotary mixer to mix well for 2h at 20 rpm.
4. Sealing of
And (3) adding 1mol/L ethanolamine 1uL to the microsphere-antibody marker obtained in the step (3), wherein the final concentration of the ethanolamine is 10mmol/L, and uniformly mixing for 1h in a rotating manner.
5. Preservation of
5.1. Washing machine
Taking a certain amount of the 4-sealed markers, performing microscopic examination, and keeping the microspheres in a monodisperse state (if the microspheres are agglomerated, performing ultrasonic treatment to the monodisperse state); centrifuging at 10000rpm for 10min, separating supernatant, resuspending the precipitate with 1mL of preservative solution, centrifuging at 10000rpm for 15min, and discarding supernatant.
5.2. Preservation of
Adding 200uL of preservation solution to resuspend and precipitate, namely, the concentration of the microspheres is 0.5%, and refrigerating and preserving at 2-8 ℃.
The concentrations and formulation methods of the reagents used in the examples were as follows:
activation buffer
19.524g MES (2- (N-morpholino) ethanesulfonic acid) and 0.05g SDS (sodium dodecyl sulfate) were weighed, a certain amount of purified water was added thereto, the pH was adjusted to 6.0, and a constant volume of 1L was obtained.
100g/L EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) solution
Weighing 10mg EDC, adding 100uL activation buffer solution, mixing well, and using as ready.
100g/L Sulfo-NHS (N-hydroxyethyl thiosuccinimide) solution
Weighing 10mg of Sulfo-NHS, adding 100uL of activation buffer solution, and fully and uniformly mixing to prepare the composition for use.
0.005% SDS solution
0.05g of SDS was weighed, and 1L of purified water was added to dissolve it sufficiently.
100mmol/L HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer solution
23.83g of HEPES was weighed, a certain amount of pure water was added, the pH was adjusted to 7.5, and a constant volume was set to 1L.
100mmol/L PB buffer
1.45g of disodium hydrogen phosphate and 0.2g of potassium dihydrogen phosphate are weighed, a certain amount of pure water is added, the pH is adjusted to 7.5, and the volume is adjusted to 100 mL.
10% Tween-20 solution
10g of Tween-20 was weighed, and 90mL of purified water was added.
1mol/L ethanolamine
0.6108g of ethanolamine was weighed out, and 100mL of purified water was added.
10mmol/L Tris-HCl buffer
1.2114g Tris (Tris hydroxymethyl aminomethane) was weighed, a certain amount of purified water was added, the pH was adjusted to 8.5, and a constant volume was set to 1L.
Preserving fluid
0.5g BSA, 0.5g Tween-20 and 0.05g ProClin300 were weighed, 100mL10mmol/L Tris-HCl buffer (pH8.5) was added, and the pH was adjusted to 8.5.
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. A coupling method of carboxyl latex microspheres and antibodies is characterized by comprising the following steps:
s1, fully mixing a stock solution of carboxyl latex microspheres with the mass fraction of 4% with an activation buffer solution according to the volume ratio of 1:15-25, centrifuging, discarding the supernatant, adding the activation buffer solution with the volume of 4 times that of the stock solution of the carboxyl latex microspheres into the precipitate, mixing again, performing ultrasonic treatment until the carboxyl latex microspheres in the solution are in a monodisperse state to obtain a microsphere suspension, and storing the microsphere suspension at 4 ℃ for later use;
s2, quickly and fully mixing 100g/L EDC aqueous solution, 100g/L Sulfo-NHS aqueous solution and the microsphere suspension according to the volume ratio of 0.8-1.2:5.5-6.5:65-75, and reacting for 30min to obtain a first mixed solution;
s3, performing ultrasonic treatment on the first mixed solution until the carboxyl latex microspheres in the first mixed solution are in a monodisperse state, performing centrifugal washing to obtain a precipitation solution, adding Tween-20 with the mass fraction of 10% into the precipitation solution, uniformly mixing to obtain an activated microsphere solution, and storing the activated microsphere solution at 4 ℃ for later use, wherein the mass fraction of the Tween-20 in the activated microsphere solution is 0.1-0.2%;
s4, mixing 0.1-1 ug/mu L of antibody solution with the activated microsphere solution according to the volume ratio of 3-4:1, and carrying out coupling reaction for more than 2h to obtain a microsphere-antibody marker solution;
s5, adding 1mol/L ethanolamine solution into the microsphere-antibody marker solution, and uniformly mixing in a rotating manner to obtain a third mixed solution, wherein the concentration of ethanolamine in the third mixed solution is 5-20 mmol/L;
s6, carrying out ultrasonic and centrifugal washing on the third mixed solution to obtain a microsphere-antibody marker precipitate, and adding a preservation solution into the microsphere-antibody marker precipitate for heavy suspension to obtain carboxyl latex microsphere-antibody marker heavy suspension containing 0.5% of mass fraction.
2. The method for coupling carboxyl latex microspheres and antibodies according to claim 1, wherein the step S1 is preceded by the steps of: and (3) carrying out ultrasonic treatment on the carboxyl latex microsphere liquid for 3-10 min.
3. The method for coupling carboxyl latex microspheres and antibodies according to claim 1, wherein the operation of centrifuging and washing in step S3 to obtain a precipitation solution comprises steps S301-302:
s301, performing first centrifugation on the second mixed solution after ultrasonic treatment at 8000-12000rpm for 10-20min, discarding the supernatant, and re-suspending the precipitate with 1mL of SDS solution to obtain a first re-suspension solution, wherein the mass fraction of the SDS solution is 0.005%;
s302, centrifuging the first resuspension solution for the second time at 8000-12000rpm for 10-20min, discarding the supernatant, adding purified water with the volume consistent with that of the stock solution of the carboxyl latex microspheres to carry out resuspension precipitation to obtain a second resuspension solution, and carrying out ultrasonic treatment on the second resuspension solution until the carboxyl latex microspheres in the second resuspension solution are in a monodisperse state to obtain the precipitation solution.
4. The method for coupling carboxyl latex microspheres and antibodies according to claim 1, wherein the step S6 of centrifugal washing to obtain the microsphere-antibody label precipitate comprises the steps S601-602:
s601, carrying out first centrifugation on the third mixed solution after the ultrasonic treatment at 8000-12000rpm for 10-20min, discarding the supernatant, and re-suspending the precipitate with 1mL of preservation solution to obtain a third suspension;
s602, carrying out second centrifugation on the third suspension at 8000-.
5. The method for coupling carboxyl latex microspheres and antibodies according to claim 4, further comprising the step of storing the carboxyl latex microsphere-antibody marker resuspension at 4 ℃ under refrigeration after step S6.
6. The method for coupling carboxyl latex microspheres and antibodies according to claim 1, wherein the antibody solution is prepared from an antibody stock solution and an antibody buffer solution, and the antibody buffer solution is 100mmol/L HEPES buffer solution or 100mmol/L PB buffer solution.
7. The method for coupling carboxyl latex microspheres and antibodies according to claim 1, wherein the pH of the activation buffer is 6, and the activation buffer comprises 19-20g MES and 0.001-1g SDS per 1L of the activation buffer.
8. The method for coupling carboxyl latex microspheres and antibodies according to claim 1, wherein the pH of the preservation solution is 8.5, and the preservation solution is prepared by adding 0.5g BSA, 0.5g Tween-20 and 0.05g ProClin300 into 100mL Tris-HCl buffer solution of 10 mmol/L.
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