CN111879921A - Fluorescent microsphere of coupled antibody and preparation method and application thereof - Google Patents
Fluorescent microsphere of coupled antibody and preparation method and application thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
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Abstract
The invention discloses an antibody-coupled fluorescent microsphere and a preparation method and application thereof, wherein a fluorescent microsphere solution, NHS (NHS) and EDC (EDC) solutions are subjected to an activation reaction under the condition of ultrasonic waves, and then the activated fluorescent microsphere solution is obtained through centrifugation, separation, dilution and dispersion; carrying out coupling reaction on the activated fluorescent microsphere solution and the antibody solution under the ultrasonic condition to obtain a coupling reaction solution; carrying out closed reaction on the coupling reaction solution and a closed solution under the ultrasonic condition to obtain a closed reaction solution; centrifuging and separating the closed reaction solution, re-dissolving and uniformly mixing to obtain fluorescent microspheres coupled with the antibody; the ultrasonic conditions are as follows: the ultrasonic frequency is 20 KHZ-80 KHZ, a multi-time ultrasonic mode is adopted, and interval time exists between two adjacent times of ultrasonic, wherein the single ultrasonic time is 3-10 s, the interval time is 3-10 s, and the total ultrasonic time is 3 min-30 min. The reaction coupling efficiency is effectively improved, and the fluorescent microspheres of the coupled antibody have good uniformity and stability.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antibody-coupled fluorescent microsphere and a preparation method and application thereof.
Background
The immunochromatography detection technology is developed based on an immunolabeling technology and a chromatography technology and can be used for detecting antigen and antibody.
The time-resolved fluorescence immunochromatographic test paper is an important detection tool in the immunochromatographic detection technology. The time-resolved fluorescent microspheres and corresponding antigen-antibody are labeled, and accurate quantitative detection of the antigen-antibody to be detected is realized through specific signals of the time-resolved fluorescent microspheres based on specific immunological reaction of the antigen-antibody.
The conventional labeling of the time-resolved fluorescent microspheres is based on EDC/NHS and other activated fluorescent microspheres, and provides activation of the microspheres and a coupling reaction environment of the microspheres and an antibody through vortex/oscillation and other uniform mixing operations. Generally speaking, through uniform mixing operations such as vortex/oscillation, coupling of the microspheres and the antigen antibody requires 2 to 24 hours, in the reaction process, regular operation of vortex/oscillation equipment often occurs, so that the reaction solution regularly contacts with the wall of a container, non-uniform adsorption of the microspheres and the antigen antibody with the wall of the container is caused, coupling efficiency of the fluorescent microspheres and the antibody is low, time consumption is too long, and stability of the prepared fluorescent microspheres of the coupled antibody is poor.
Therefore, how to develop a preparation method of fluorescent microspheres coupled with antibodies, which has the advantages of short time consumption, high coupling efficiency of the fluorescent microspheres and the antibodies and good stability, is a technical problem to be solved urgently.
Disclosure of Invention
The invention aims to provide an antibody-coupled fluorescent microsphere and a preparation method and application thereof. The fluorescent microsphere solution coupled with the antibody is used for preparing a fluorescent immunochromatographic kit, and the risk of activity reduction of the antigen antibody in the reaction process is reduced, and the reaction signal intensity is improved, so that the detection sensitivity and stability of the fluorescent immunochromatographic kit are improved, and the production cost is reduced.
In order to achieve the above object, the present invention provides a method for preparing an antibody-conjugated fluorescent microsphere, the method comprising:
obtaining a fluorescent microsphere solution, an NHS solution, an EDC solution and a crosslinking reaction solution;
carrying out activation reaction on the fluorescent microsphere solution, the NHS solution and the EDC solution under a first ultrasonic condition, then carrying out centrifugation and/or solid-liquid separation to obtain a first solid, diluting the first solid by using the crosslinking reaction solution, and then dispersing to obtain an activated fluorescent microsphere solution;
obtaining an antibody solution; uniformly mixing the activated fluorescent microsphere solution and the antibody solution, and then carrying out coupling reaction under the second ultrasonic condition to obtain a coupling reaction solution;
obtaining sealing liquid; uniformly mixing the coupling reaction solution and the sealing solution, and then carrying out sealing reaction under a third ultrasonic condition to obtain a sealing reaction solution;
obtaining a redissolution; centrifuging and/or performing solid-liquid separation on the closed reaction solution to obtain a second solid, and uniformly mixing the second solid after re-suspending the second solid by using the re-dissolving solution to obtain fluorescent microspheres coupled with the antibody;
the first ultrasonic condition, the second ultrasonic condition, and the third ultrasonic condition are all: the ultrasonic frequency is 20 KHZ-80 KHZ, a multi-time ultrasonic mode is adopted, and interval time exists between two adjacent times of ultrasonic, wherein the single ultrasonic time is 3-10 s, the single interval time is 3-10 s, and the total ultrasonic time is 3 min-30 min.
Further, the total time of the ultrasound in the first ultrasonic wave is 3min to 10 min.
Further, the total time of the ultrasound in the second ultrasonic wave is 10min to 20 min.
Further, the total time of the ultrasound in the third ultrasonic condition is 5min to 15 min.
Further, the concentration of the NHS solution is 1 ug/mL-500 ug/mL; the concentration of the EDC solution is 1 ug/mL-500 ug/mL; the mass fraction of the fluorescent microspheres in the fluorescent microsphere solution is 0.05-1%; the surface of the fluorescent microsphere contains carboxyl or amino; the particle size of the fluorescent microsphere is 50nm-500 nm.
Further, the crosslinking reaction solution comprises a first buffer solution with the concentration of 0.01-0.2 mol/L, and the first buffer solution comprises one or more of phosphate buffer solution, HEPES buffer solution, carbonate buffer solution and MES buffer solution.
Further, the volume ratio of the added sealing liquid to the fluorescent microsphere solution is 1: 5-50; the blocking solution comprises glycine solution and/or BSA solution.
Further, the redissolution solution comprises a second buffer solution with the concentration of 0.01-0.2 mol/L, and the second buffer solution comprises one or more of phosphate buffer solution, HEPES buffer solution, carbonate buffer solution and MES buffer solution; the reconstitution solution further includes one or more of a surfactant, a polyol, and a protein.
The invention also provides the fluorescent microsphere of the coupled antibody prepared by the method.
The invention also provides application of the fluorescent microsphere coupled with the antibody in preparation of a fluorescence immunochromatographic kit.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
according to the fluorescent microsphere of the coupled antibody and the preparation method thereof provided by the invention, the fluorescent microsphere solution of the coupled antibody has good stability, can be stably stored for a long time, has high coupling efficiency, effectively improves the reaction coupling efficiency, and obviously reduces the time required by the reaction of labeling the antigen and the antibody by the fluorescent microsphere. The fluorescent microsphere solution coupled with the antibody is used for preparing a fluorescent immunochromatographic kit, and the risk of activity reduction of the antigen antibody in the reaction process is reduced, and the reaction signal intensity is improved, so that the detection sensitivity and stability of the fluorescent immunochromatographic kit are improved, and the production cost is reduced.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a flow chart of a method for preparing fluorescent microspheres coupled with antibodies according to the present invention;
FIG. 2 is a diagram illustrating the structure of a fluorescence immunochromatographic kit according to an embodiment of the present invention; 1. a base plate; 11. a first end portion; 12. a second end portion; 2. a sample application hole; 3. a sample pad; 4. a fluorescent pad; 5. a nitrocellulose membrane; 51. detecting lines; 52. a quality control line; 6. an absorbent pad.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method. The terms "first", "second", and the like in the present invention do not denote any order, and they may be understood as nouns.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
according to an exemplary embodiment of the present invention, there is provided a method for preparing fluorescent microspheres coupled with antibodies, as shown in fig. 1, including:
s1, obtaining a fluorescent microsphere solution, an NHS solution, an EDC solution and a crosslinking reaction solution;
carrying out activation reaction on the fluorescent microsphere solution, the NHS solution and the EDC solution under a first ultrasonic condition, then carrying out centrifugation and/or solid-liquid separation to obtain a first solid, diluting the first solid by using the crosslinking reaction solution, and then dispersing to obtain an activated fluorescent microsphere solution;
s2, obtaining an antibody solution; uniformly mixing the activated fluorescent microsphere solution and the antibody solution, and then carrying out coupling reaction under the second ultrasonic condition to obtain a coupling reaction solution;
s3, obtaining sealing liquid; uniformly mixing the coupling reaction solution and the sealing solution, and then carrying out sealing reaction under a third ultrasonic condition to obtain a sealing reaction solution;
s4, obtaining a redissolution; and (3) carrying out centrifugation and/or solid-liquid separation on the closed reaction solution to obtain a second solid, and carrying out re-suspension on the second solid by using the re-dissolving solution and then uniformly mixing to obtain the fluorescent microspheres coupled with the antibody.
The first ultrasonic condition, the second ultrasonic condition, and the third ultrasonic condition are all: the ultrasonic frequency is 20 KHZ-80 KHZ, a multi-time ultrasonic mode is adopted, and interval time exists between two adjacent times of ultrasonic, wherein the single ultrasonic time is 3-10 s, the interval time is 3-10 s, and the total ultrasonic time is 3 min-30 min.
In the present embodiment, the first and second embodiments are described,
the fluorescent microspheres are: can be polystyrene microspheres doped/modified with fluorescent signals such as fluorescein, europium ions and the like. The surface of the fluorescent microsphere contains carboxyl or amino; the particle size of the fluorescent microsphere is between 50nm and 500 nm; the mass concentration of the fluorescent microspheres can be 1% (W/W) to 0.05% (W/W); the buffer solution may be washed with an activation buffer before use.
The activating solution: the concentration of NHS is between 1ug/ml and 500ug/ml, and the NHS can be dissolved by pure water, ethanol or an activating solution; the concentration of EDC is between 1ug/ml and 500ug/ml, and the EDC can be dissolved by pure water, ethanol or activating solution.
The activation buffer: MES solution, the concentration of MES solution is between 0.01M and 0.2M.
The antigen/antibody: the final concentration is between 10ug/ml and 300 ug/ml. The antibody can be interleukin 6 antibody, procalcitonin antibody, D dimer antibody, cardiac troponin antibody and the like;
the crosslinking reaction solution: phosphate buffer, HEPES buffer, carbonate buffer, MES buffer, etc.; the concentration of the crosslinking reaction solution is between 0.01M and 0.2M; contains surfactant, generally including but not limited to triton X-100, Tween 20, brij-35, OP-10, Tetronic1307, etc.;
the sealing agent is: a solution containing 10% BSA, or a solution of 10% BSA and 10% glycine. The solution may be in an aqueous or buffer matrix. The buffer solution can be phosphate buffer solution, HEPES buffer solution, carbonate buffer solution, MES buffer solution and the like;
the redissolution solution: it may be phosphate buffer, HEPES buffer, carbonate buffer, MES buffer, etc. The concentration of the redissolution solution is between 0.01M and 0.2M. The reconstitution solution may contain a concentration of surfactant. The surfactant generally includes, but is not limited to, Triton X-100, Tween 20, brij-35, OP-10, Tetronic1307, and the like. The concentration of the surfactant is 0.02% (W/W) to 1% (W/W). The reconstituted solution may contain a concentration of polyhydroxy compounds including, but not limited to, sucrose, trehalose, HPMC, chitosan, and the like. The reconstitution solution may contain a concentration of protein including, but not limited to, BSA, casein, gelatin, skim milk powder, etc.
The preparation method of the fluorescent microsphere coupled with the antibody is based on the following principle:
the invention adopts the activation reaction, the coupling reaction and the sealing reaction under the ultrasonic condition, improves the full mixing and collision probability of the microspheres and the antigen antibody to be marked, and also effectively reduces the reaction heterogeneity caused by the aggregation of the microspheres in the reaction process, thereby obviously improving the coupling efficiency and the coupling proportion of the fluorescent microspheres and the antigen antibody. Meanwhile, the fluorescent microspheres and the antigen antibody are fully reacted, so that the conventional coupling method of the labeled fluorescent microspheres and the antigen antibody, which corresponds to the compounding of the antigen antibody, can be effectively improved. The negative influence of the heterogeneity of the microsphere solution on the reaction and the risk of the activity reduction of the antigen and the antibody in a long-time unstable environment in the process of overlong reaction time are avoided.
In the prior art, activation reaction, coupling reaction and blocking reaction are usually carried out by adopting a rotary mixing mode, and the defects that the coupling efficiency of the fluorescent microsphere and the antibody is low, the time consumption is too long, and the uniformity and the stability of the prepared fluorescent microsphere of the coupled antibody are poor exist.
Although the prior art also has ultrasound, the ultrasound only plays a role of mixing, and mainly the microspheres are agglomerated after the coupling is finished, and the ultrasound is carried out once in order to break up the agglomerated materials.
The invention firstly provides a technical scheme of carrying out activation reaction, coupling reaction and sealing reaction under the ultrasonic condition, the ultrasonic aim is to promote the reaction and accelerate the coupling reaction of the antibody and the microsphere, the time in the process is reduced, the influence of the change of the protein along with the time is less, and the process is more controllable.
This is not the case in the prior art because it is difficult for those skilled in the art to perform the activation reaction, coupling reaction, and blocking reaction under ultrasonic conditions, which usually results in microsphere aggregation, antibody inactivation, and poor coupling of antibody and microsphere.
Therefore, how to control the ultrasonic condition to enable the activation reaction, the coupling reaction and the sealing reaction to be better carried out, and effectively improving the coupling efficiency of the reaction becomes the difficult point of the technical scheme of the invention. The inventor finds through experiments that the technical problem can be solved well by adopting the following ultrasonic conditions:
the first ultrasonic condition, the second ultrasonic condition and the third ultrasonic condition are all as follows: the ultrasonic frequency is 20 KHZ-80 KHZ, the ultrasonic comprises multiple times of ultrasonic, the interval is formed between two adjacent times of ultrasonic, the ultrasonic time is 3-10 s each time, the interval time is 3-10 s, and the total ultrasonic time is 3-30 min. The protein can play a role in promoting reaction within the parameter range, and the coupling effect of the protein is good.
If the ultrasonic frequency is lower than 20KHZ, the ultrasonic time is less than 3s each time, the coupling effect is poor due to the interval time of less than 3s, the reaction promotion effect cannot be realized, the activation, coupling and sealing of the microspheres are insufficient, and the protein concentration of the microsphere solution tested after centrifugation is lower; the reaction strength of the prepared test paper is obviously weak.
If the ultrasonic frequency is higher than 80KHZ, the ultrasonic time is longer than 10s each time, the interval time is longer than 10s, the protein is easy to change, the coupling effect of the protein is poor, the protein is inactivated, the protein concentration of the microsphere solution is low after centrifugation, the test paper is prepared, and the reaction strength is remarkably weak.
Preferably, the ultrasonic conditions suitable for the activation reaction, the coupling reaction and the blocking reaction are further refined, specifically:
the total ultrasonic time in the first ultrasonic wave is 3min to 10min (optimally 5 min); this range is found by the inventors of the present invention through experimental exploration to more preferably play a role in promoting the activation reaction.
The total ultrasonic time in the second ultrasonic wave is 10 min-20 min (optimally 15 min); the range is found by the inventor through experimental exploration to better play a role in promoting the coupling reaction.
The total ultrasonic time in the third ultrasonic condition is 5-15 min (preferably 10 min). The range is found by the inventor through experimental exploration to better play the role of promoting the blocking reaction.
According to another exemplary embodiment of the present invention, there is provided the antibody-coupled fluorescent microsphere prepared by the method, wherein the antibody-coupled fluorescent microsphere solution has good uniformity and high coupling efficiency: the method is adopted to carry out ultrasonic activation for 5min in the activation stage, the activation is 20min in the non-ultrasonic method in the prior art, the efficiency of coupling the microspheres with the antibody is the same, the activation degree which can be realized only by the non-ultrasonic method for 20min can be achieved after 5min of ultrasonic activation, and the efficiency of activating the microspheres by the ultrasonic method is obviously improved. Coupling efficiency, if measured in terms of time, is the same ratio of the time shortened by the process of the invention (15min) to the time required by the prior art (20 min).
In the coupling stage, the reaction strength of the coupled microsphere antigen antibody is close to that of the coupled microsphere antigen antibody in 15min by an ultrasonic method and a conventional non-ultrasonic method for 4 hours, which shows that the protein coupling efficiency can reach the effect of the non-ultrasonic method for 4 hours in 15min by ultrasonic, and the efficiency of the coupling of the ultrasonic microsphere antibody is obviously improved.
And the fluorescent microsphere solution coupled with the antibody is prepared into the reagent strip, so that the sensitivity is higher and the stability is better.
According to another exemplary embodiment of the present invention, the present invention further provides a use of the antibody-conjugated fluorescent microsphere in the preparation of a fluorescence immunochromatographic kit:
as shown in fig. 2, the fluorescence immunochromatographic kit comprises:
base plate 1 said base plate having a first end 11 and a second end 12;
the sample adding hole 2, the sample pad 3, the fluorescent pad 4, the nitrocellulose membrane 5 and the absorbent pad 6 are sequentially arranged along the direction from the first end part to the second end part of the bottom plate 1;
the fluorescent microspheres of the coupled antibody are sprayed on the fluorescent pad 4;
the nitrocellulose membrane 5 is sequentially provided with a detection line 51 and a quality control line 52 along the direction from the first end part 11 to the second end part 12 of the bottom plate, wherein an immobilized antibody is immobilized on the detection line 51, and a goat-anti-mouse secondary antibody or a goat-anti-mouse polyclonal antibody is immobilized on the quality control line 52.
Diluting the fluorescent microspheres coupled with the antibody to a proper volume, spraying the diluted fluorescent microspheres on a fluorescent pad 4, drying the fluorescent pad at 45 ℃, matching the pre-coated NC membrane, the sample pad and other components, and assembling to prepare the fluorescent immunochromatographic kit.
The fluorescence immunochromatographic kit can be prepared for various antibodies, including but not limited to:
when the antibody solution is an interleukin 6 antibody, preparing fluorescent microspheres coupled with the interleukin 6 antibody, spraying the fluorescent microspheres coupled with the interleukin 6 antibody on the fluorescent pad 4, solidifying antibodies on the detection line 51, and solidifying goat-anti-mouse secondary antibodies or goat-anti-mouse polyclonal antibodies on the quality control line 52;
d dimer fluorescence immunochromatography kit:
when the antibody solution is a D dimer antibody, preparing fluorescent microspheres coupled with the D dimer antibody, spraying the fluorescent microspheres coupled with the D dimer antibody on the fluorescent pad 4, curing an antibody on the detection line 51, and curing a goat-anti-mouse secondary antibody or a goat-anti-mouse polyclonal antibody on the quality control line 52;
the fluorescence immunochromatography kit is high in sensitivity and good in stability, and specifically comprises the following components:
the sensitivity of the interleukin 6 fluorescence immunochromatographic kit is 3.00pg/mL, and the kit can be stably stored for 18 months.
The sensitivity of the D dimer fluorescence immunochromatography kit is 0.20mg/L, and the D dimer fluorescence immunochromatography kit can be stably stored for 18 months.
According to the fluorescent microsphere of the coupled antibody, the preparation method and the application thereof, provided by the invention, the reaction coupling efficiency is effectively improved, the time required by the reaction of the fluorescent microsphere labeled with the antigen antibody is obviously reduced, and the prepared fluorescent microsphere of the coupled antibody is good in uniformity and stability. The fluorescent microspheres coupled with the antibody are used for preparing a fluorescence immunochromatographic kit, and the risk of activity reduction of the antigen antibody in the reaction process is reduced, and the reaction signal intensity is improved, so that the detection sensitivity and stability of the fluorescence immunochromatographic kit are improved, and the production cost is reduced.
The following will describe in detail the preparation method of the antibody-conjugated fluorescent microspheres of the present application with reference to examples, comparative examples and experimental data.
EXAMPLES 1-5 preparation of fluorescent microspheres conjugated with Interleukin-6 antibody Using the method of the present invention
S1, obtaining a fluorescent microsphere solution: diluting fluorescent microspheres with particle size of 200nm to 0.1% (W/W) with 0.01M MES pH6.0 activation buffer, and the volume is 1 ml;
NHS solution: NHS is dissolved to 10mg/ml by purified water, and the addition amount of the NHS solution is 10 ug/ml;
EDC solution: EDC is dissolved to 10mg/ml by purified water, and the adding amount of EDC solution is 20 ug/ml;
crosslinking reaction solution: 0.01M PB, pH7.4 crosslinking reaction solution;
s2, carrying out activation reaction on the fluorescent microsphere solution, the NHS solution and the EDC solution under the condition of first ultrasonic wave, then carrying out first centrifugation and first solid-liquid separation to obtain a first solid, diluting the first solid by using the crosslinking reaction solution, and then dispersing to obtain an activated fluorescent microsphere solution;
s3, obtaining antibody solution: the antibody solution is interleukin 6 antibody, the concentration of the interleukin 6 antibody is 1mg/ml, and the total amount of the antibody is 100 ug;
uniformly mixing the activated fluorescent microsphere solution and the antibody solution, and then carrying out coupling reaction under the second ultrasonic condition to obtain a coupling reaction solution;
s4, obtaining a sealing liquid: taking 100ul of sealant; the blocking agent is 10% BSA solution;
uniformly mixing the coupling reaction solution and the sealing solution, and then carrying out sealing reaction under a third ultrasonic condition to obtain a sealing reaction solution;
s5, obtaining a redissolution: 0.01M pH 8.0 tris-HCl, 1% sucrose and 0.1% Triton X-100. (ii) a
And carrying out second centrifugation and second solid-liquid separation on the closed reaction solution to obtain a second solid, and carrying out re-suspension on the second solid by using the re-dissolving solution and then uniformly mixing to obtain the fluorescent microspheres coupled with the interleukin 6 antibody.
The first ultrasonic condition, the second ultrasonic condition, and the third ultrasonic condition are shown in table 1.
Meanwhile, the fluorescent microspheres coupled with the interleukin 6 antibody obtained in the embodiment 1 are diluted by 5ml, sprayed on a fluorescent pad, dried at 45 ℃, matched with pre-coated components such as an NC membrane, a sample pad and the like, and assembled to prepare a detection card, namely the interleukin 6 fluorescent immunochromatography kit.
Example 6 preparation of fluorescent microspheres coupled with D dimer antibody Using the method of the present invention
S1, obtaining a fluorescent microsphere solution: diluting fluorescent microspheres with particle size of 300nm to 0.1% (W/W) with 0.01M MES pH6.0 activation buffer solution, and the volume is 1 ml; vortex, mix well, then 15000rpm, high speed centrifugation for 15min, remove the supernatant, precipitate with 0.01M MES pH6.0 activation buffer diluted to 1ml
NHS solution: NHS is dissolved to 10mg/ml by purified water;
EDC solution: EDC is dissolved to 10mg/ml by purified water, and the adding amount of EDC solution is 30 ug/ml;
crosslinking reaction solution: 0.01M PB, pH7.4 crosslinking reaction solution;
s2, carrying out activation reaction on the fluorescent microsphere solution, the NHS solution and the EDC solution under the condition of first ultrasonic wave, then carrying out first centrifugation and first solid-liquid separation to obtain a first solid, diluting the first solid by using the crosslinking reaction solution, and then dispersing to obtain an activated fluorescent microsphere solution;
s3, obtaining antibody solution: the antibody solution is a D dimer antibody;
uniformly mixing the activated fluorescent microsphere solution and the antibody solution, and then carrying out coupling reaction under the second ultrasonic condition to obtain a coupling reaction solution;
s4, obtaining a sealing liquid: an aqueous solution containing 10% BSA and 10% glycine;
uniformly mixing the coupling reaction solution and the sealing solution, and then carrying out sealing reaction under a third ultrasonic condition to obtain a sealing reaction solution;
s5, obtaining a redissolution: 0.01M, pH 8.0 tris-HCL, 1% sucrose and 0.1% Triton X-100;
and carrying out second centrifugation and second solid-liquid separation on the closed reaction solution to obtain a second solid, and carrying out re-suspension on the second solid by using the re-dissolving solution and then uniformly mixing to obtain the fluorescent microspheres coupled with the D dimer antibody.
The first ultrasonic condition, the second ultrasonic condition, and the third ultrasonic condition are shown in table 1.
And (3) diluting 5ml of the fluorescent microspheres coupled with the D dimer antibody, spraying the fluorescent microspheres on a fluorescent pad, drying the fluorescent pad at 45 ℃, matching pre-coated components such as an NC (numerical control) membrane, a sample pad and the like, and assembling the components to prepare a detection card, namely the D dimer fluorescence immunochromatographic kit.
Comparative example 1-comparative example 6 fluorescent microspheres coupled with interleukin 6 antibody
Comparative example 1 to comparative example 6 the procedure of examples 1 to 5 was the same except that the first ultrasonic wave condition, the second ultrasonic wave condition and the third ultrasonic wave condition were different from those of examples 1 to 6. The first ultrasonic condition, the second ultrasonic condition, and the third ultrasonic condition are shown in table 1.
TABLE 1 Process parameters for the examples and comparative examples
Comparative example 7 preparation of fluorescent microsphere coupled with Interleukin 6 antibody by Prior Art method
(1) Preparing a fluorescent microsphere solution: diluting fluorescent microspheres with particle size of 200nm to 0.1% (W/W) with 0.01M MES pH6.0 activation buffer, and the volume is 1 ml;
(2) dissolving NHS with purified water to 10mg/ml, quickly adding the solution into the fluorescent microsphere solution, and uniformly mixing the solution in a vortex mode, wherein the adding amount of the NHS solution is 10 ug/ml;
(3) dissolving EDC with purified water to 10mg/ml, then rapidly adding into the fluorescent microsphere solution, and uniformly mixing by vortex, wherein the addition amount of the EDC solution is 20 ug/ml;
(4) placing the fluorescent microsphere solution in a shaking table for activation reaction, controlling the rotating speed at 220rpm, activating for 30min, and controlling the temperature at 30 ℃;
(5) centrifuging at 15000rpm for 20min, removing supernatant, diluting the precipitate with 0.01M PB, pH7.4 crosslinking reaction solution to 1ml, and ultrasonic dispersing for 2 min; the ultrasonic reaction parameters are 5 seconds of ultrasonic treatment/5 seconds of interval, and the ultrasonic treatment lasts for 12 cycles, namely 2 min.
(6) Adding the fluorescent microsphere solution into an interleukin 6 antibody solution, wherein the concentration of the interleukin 6 antibody is 1mg/ml, the total amount of the antibody is 100ug, and uniformly mixing by vortex;
(7) placing the fluorescent microsphere solution in a shaking table for coupling reaction, controlling the rotating speed at 220rpm, controlling the coupling time at 8 hours and controlling the temperature at 30 ℃.
(8) Adding 100ul of sealant into the fluorescent microsphere solution, and uniformly mixing by vortex; the blocking agent was 10% BSA solution.
(9) Placing the fluorescent microsphere solution in a shaking table for sealing reaction, controlling the rotating speed at 220rpm, controlling the sealing time at 2 hours and controlling the temperature at 30 ℃.
(10) Centrifuging at 15000rpm for 20min, removing supernatant, resuspending the precipitate to 1ml with 0.01M tris-HCL reconstituted solution with pH 8.0, and mixing by vortex; the reconstituted solution contained 1% sucrose and 0.1% Triton X-100.
(11) Diluting 5ml of the redissolved fluorescent microsphere solution;
(12) and spraying the fluorescent microspheres on the bonding pad, drying at 45 ℃, matching the pre-coated NC membrane, the sample pad and other components, and assembling to prepare the detection card, namely the interleukin 6 fluorescence immunochromatography kit.
Comparative example 8 preparation of fluorescent microspheres coupled with D dimer antibody by Prior Art Process
(1) Preparing a fluorescent microsphere solution: diluting fluorescent microspheres with particle size of 300nm to 0.1% (W/W) with 0.01M MES pH6.0 activation buffer, and the volume is 1 ml; vortex, mix evenly, then 15000rpm, high speed centrifugation for 15min, remove the supernatant, precipitate with 0.01M MES pH6.0 activation buffer diluted to 1 ml;
(2) dissolving NHS with purified water to 10mg/ml, quickly adding the solution into the fluorescent microsphere solution, and uniformly mixing the solution in a vortex mode, wherein the adding amount of the NHS solution is 15 ug/ml;
(3) dissolving EDC with purified water to 10mg/ml, rapidly adding into fluorescent microsphere solution, mixing by vortex, and adding EDC solution at 30ug/ml
(4) Placing the fluorescent microsphere solution in a shaking table for activation reaction, controlling the rotating speed at 220rpm, activating for 30min, and controlling the temperature at 30 ℃;
(5) centrifuging at 15000rpm for 15min, removing supernatant, diluting the precipitate with 0.01MPB, pH7.4 crosslinking reaction solution to 1ml, and ultrasonically dispersing for 2 min; the ultrasonic reaction parameters are 5 seconds of ultrasonic treatment/5 seconds of interval, and the ultrasonic treatment lasts for 12 cycles, namely 2 min.
(6) Adding the fluorescent microsphere solution into D dimer antibody solution, wherein the concentration of the D dimer antibody is 1mg/ml, the total amount of the antibody is 50ug, and uniformly mixing by vortex
(7) Placing the fluorescent microsphere solution in a shaking table for coupling reaction, controlling the rotating speed at 220rpm, controlling the coupling time at 8 hours and controlling the temperature at 30 ℃.
(8) Adding 100ul of sealant into the fluorescent microsphere solution, and uniformly mixing by vortex; the blocking agent is an aqueous solution containing 10% BSA and 10% glycine.
(9) Placing the fluorescent microsphere solution in a shaking table for sealing reaction, controlling the rotating speed at 220rpm, controlling the sealing time at 2 hours and controlling the temperature at 30 ℃.
(10) Centrifuging at 15000rpm for 15min, removing supernatant, resuspending the precipitate to 1ml with 0.01M tris-HCL reconstituted solution with pH 8.0, and mixing by vortex; the reconstituted solution contained 1% sucrose and 0.1% Triton X-100.
(11) Diluting 5ml of the redissolved fluorescent microsphere solution;
(12) and spraying the fluorescent microspheres on the bonding pad, drying at 45 ℃, matching the pre-coated NC membrane, the sample pad and other components, and assembling to prepare the detection card, namely the D dimer fluorescence immunochromatography kit.
Test example 1 stability of antibody-conjugated fluorescent microsphere solution
The antibody-conjugated fluorescent microsphere solutions prepared in examples 1 to 6 and comparative examples 1 to 6 were stored in a refrigerator at 4 ℃ for 7 days, and the state of the antibody-fluorescent microsphere conjugate was visually observed, and the results are shown in Table 2.
TABLE 2 fluorescent microsphere Performance of groups of conjugated antibodies
| Group of | Stability of |
| Example 1 | After 7 days no precipitate appeared |
| Example 2 | After 5 days no precipitate appeared |
| Example 3 | After 6 days no precipitate appeared |
| Example 4 | After 6 days no precipitate appeared |
| Example 5 | After 7 days no precipitate appeared |
| Example 6 | After 7 days no precipitate appeared |
| Comparative example 1 | After 3 days, a partial precipitate was visible at the bottom |
| Comparative example 2 | After 3 days, a partial precipitate was visible at the bottom |
| Comparative example 3 | After 3 days, a partial precipitate was visible at the bottom |
| Comparative example 4 | After 2 days, a partial precipitate was visible at the bottom |
| Comparative example 5 | After 1 day, a partial precipitate was visible at the bottom |
| Comparative example 6 | After 1 day, a partial precipitate was visible at the bottom |
From the data in table 2, it can be seen that:
comparative example 1: the ultrasonic frequency is less than 20KHZ, and the stability is poor; the activation, coupling and sealing of the microspheres are not sufficient, and the protein concentration of the microsphere solution is low after centrifugation; in addition, the reaction strength after the test paper is prepared is remarkably weak.
Comparative example 2: the ultrasonic frequency is higher than 80KHZ, the coupling effect of the protein is poor, the protein is inactivated, and the stability is poor; after centrifugation, the protein concentration of the test microsphere solution is lower; in addition, the reaction strength is obviously weak after the test paper is prepared.
Comparative example 3: each ultrasonic time is less than 3s, the interval time is less than 3s, and the stability is poor; the activation, coupling and sealing of the microspheres are not sufficient, and the protein concentration of the microsphere solution is low after centrifugation; in addition, the reaction strength after the test paper is prepared is remarkably weak.
Comparative example 4: each time of ultrasonic treatment is longer than 10s, the interval time is longer than 10s, the coupling effect of the protein is poor, the protein is inactivated, and the stability is poor; after centrifugation, the protein concentration of the test microsphere solution is low, and in addition, the reaction strength is remarkably weak after the test paper is prepared.
Comparative example 5: the total ultrasonic time is less than 3min, and the stability is poor; the activation, coupling and sealing of the microspheres are not sufficient, and the protein concentration of the microsphere solution is low after centrifugation; in addition, the reaction strength after the test paper is prepared is remarkably weak.
Comparative example 6: the total ultrasonic time is more than 30min, the coupling effect of the protein is poor, the protein is inactivated, and the stability is poor; after centrifugation, the protein concentration of the test microsphere solution is low, and in addition, the reaction strength is remarkably weak after the test paper is prepared.
In the embodiments 1 to 6 of the present invention, the reaction coupling efficiency is effectively improved, and the obtained fluorescent microspheres of the coupled antibody have good stability.
Test example 2: performance determination of fluorescence immunochromatography kit
The sensitivity and stability of the interleukin 6 fluorescence immunochromatographic kit prepared in the embodiment 1, the interleukin 6 fluorescence immunochromatographic kit prepared in the embodiment 6, the interleukin 6 fluorescence immunochromatographic kit prepared in the comparative example 7 and the interleukin 6 fluorescence immunochromatographic kit prepared in the comparative example 8 are detected and counted.
(1) Functional sensitivity: the minimum amount of analyte in the sample can be quantified with a specified degree of acceptable precision and accuracy. Interleukin 6 evaluation method: measuring blank samples and low-concentration samples with interleukin 6 concentration of 0.2mg/L, 0.4mg/L and 0.8mg/L, wherein each sample is measured for 10 times; d dimer evaluation method: the blank sample, D dimer concentration was determined as: 0.1mg/L, 0.25mg/L, 0.5mg/L, 1.0mg/L
Calculating the mean value, the standard deviation, the deviation and the coefficient of variation, wherein the CV is less than 10 percent and the deviation is less than 10 percent, so that the use requirement can be met;
TABLE 3 Interleukin 6 sensitivity data
As can be seen from the data in Table 3, the functional sensitivity of example 1 was 3.00pg/mL, the functional sensitivity of comparative example 7 was more than 7pg/mL, for the interleukin 6 item, the reference range was generally 1-7pg/mL, and the precision of the concentration range of 7pg/mL was important for the determination of negative and positive, so the functional sensitivity of the reagent had a great influence on the performance of the reagent
TABLE 4-D dimer sensitivity data
As can be seen from the data in Table 4, the functional sensitivity of example 6 was 0.20mg/L, the functional sensitivity of comparative example 8 was more than 0.50mg/L, the reference range was generally 0 to 0.5mg/L for the D dimer item, and the precision of the concentration range of 0.5mg/L was important for the judgment of negative and positive, so the functional sensitivity of the reagent had a great influence on the performance of the reagent.
(2) Stability: placing the reagent at 2-8 ℃ for 24 months, and observing and measuring the deviation of the third-party quality control data; the results are shown in the following table.
TABLE 5 Interleukin 6 stability data
TABLE 6-D dimer stability data
As can be seen from the above data, the fluorescence immunochromatographic kits of examples 1 and 6 are more stable and can be stably stored for 18 months, the fluorescence immunochromatographic kits of comparative examples 7 and 8 gradually decrease in measured value with the increase of sample retention time until the measured value exceeds the allowable range, and the stability is inferior to that of the fluorescence immunochromatographic kits of examples 1 and 6.
In conclusion, the fluorescent microsphere of the coupled antibody, the preparation method and the application thereof provided by the invention effectively improve the reaction coupling efficiency, remarkably reduce the time required by the reaction of the fluorescent microsphere labeled with the antigen and the antibody, and the prepared fluorescent microsphere of the coupled antibody has good uniformity and stability. The fluorescent microspheres coupled with the antibody are used for preparing a fluorescence immunochromatographic kit, and the risk of activity reduction of the antigen antibody in the reaction process is reduced, and the reaction signal intensity is improved, so that the detection sensitivity and stability of the fluorescence immunochromatographic kit are improved, and the production cost is reduced.
Finally, it should also be noted that 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.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A method for preparing fluorescent microspheres coupled with antibodies, which is characterized by comprising the following steps:
obtaining a fluorescent microsphere solution, an NHS solution, an EDC solution and a crosslinking reaction solution;
carrying out activation reaction on the fluorescent microsphere solution, the NHS solution and the EDC solution under a first ultrasonic condition, then carrying out centrifugation and/or solid-liquid separation to obtain a first solid, diluting the first solid by using the crosslinking reaction solution, and then dispersing to obtain an activated fluorescent microsphere solution;
obtaining an antibody solution; uniformly mixing the activated fluorescent microsphere solution and the antibody solution, and then carrying out coupling reaction under the second ultrasonic condition to obtain a coupling reaction solution;
obtaining sealing liquid; uniformly mixing the coupling reaction solution and the sealing solution, and then carrying out sealing reaction under a third ultrasonic condition to obtain a sealing reaction solution;
obtaining a redissolution; centrifuging and/or performing solid-liquid separation on the closed reaction solution to obtain a second solid, and uniformly mixing the second solid after re-suspending the second solid by using the re-dissolving solution to obtain fluorescent microspheres coupled with the antibody;
the first ultrasonic condition, the second ultrasonic condition, and the third ultrasonic condition are all: the ultrasonic frequency is 20 KHZ-80 KHZ, a multi-time ultrasonic mode is adopted, and interval time exists between two adjacent times of ultrasonic, wherein the single ultrasonic time is 3-10 s, the single interval time is 3-10 s, and the total ultrasonic time is 3 min-30 min.
2. The method for preparing antibody-conjugated fluorescent microspheres according to claim 1, wherein the total time of the ultrasound in the first ultrasonic wave is 3-10 min.
3. The method for preparing antibody-conjugated fluorescent microspheres according to claim 1, wherein the total time of the ultrasound in the second ultrasonic wave is 10-20 min.
4. The method for preparing antibody-conjugated fluorescent microspheres according to claim 1, wherein the total time of the ultrasound in the third ultrasonic condition is 5-15 min.
5. The method for preparing antibody-conjugated fluorescent microspheres according to claim 1, wherein the concentration of the NHS solution is 1ug/mL to 500 ug/mL; the concentration of the EDC solution is 1 ug/mL-500 ug/mL; the mass fraction of the fluorescent microspheres in the fluorescent microsphere solution is 0.05-1%; the surface of the fluorescent microsphere contains carboxyl or amino; the particle size of the fluorescent microsphere is 50nm-500 nm.
6. The method of claim 1, wherein the crosslinking reaction solution comprises a first buffer solution with a concentration of 0.01mol/L to 0.2mol/L, and the first buffer solution comprises one or more of phosphate buffer, HEPES buffer, carbonate buffer, and MES buffer.
7. The method for preparing antibody-conjugated fluorescent microspheres according to claim 1, wherein the ratio of the volume of the added blocking solution to the volume of the fluorescent microsphere solution is 1: 5-50; the blocking solution comprises glycine solution and/or BSA solution.
8. The method for preparing antibody-coupled fluorescent microspheres according to claim 1, wherein the reconstitution solution comprises a second buffer solution with a concentration of 0.01mol/L to 0.2mol/L, and the second buffer solution comprises one or more of phosphate buffer solution, HEPES buffer solution, carbonate buffer solution, and MES buffer solution; the reconstitution solution further includes one or more of a surfactant, a polyol, and a protein.
9. An antibody-conjugated fluorescent microsphere prepared according to any one of claims 1 to 8.
10. Use of the antibody-conjugated fluorescent microspheres of claim 9 for the preparation of a fluorescence immunochromatographic kit.
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