CN111077315A - Coupling method of fluorescent microspheres and antibody - Google Patents
Coupling method of fluorescent microspheres and antibody Download PDFInfo
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- CN111077315A CN111077315A CN201911350918.8A CN201911350918A CN111077315A CN 111077315 A CN111077315 A CN 111077315A CN 201911350918 A CN201911350918 A CN 201911350918A CN 111077315 A CN111077315 A CN 111077315A
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Abstract
The embodiment of the invention discloses a coupling method of fluorescent microspheres and antibodies, which comprises the following steps: activating the fluorescent microspheres to obtain activated fluorescent microspheres; adding MES buffer solution containing glycerol into the activated fluorescent microspheres, ultrasonically mixing, adding antibody solution, ultrasonically reacting, and rotationally mixing in a dark place; after the reaction is finished, adding confining liquid into the precipitate, ultrasonically mixing uniformly, rotating and mixing uniformly in a dark place, centrifuging, removing supernatant, continuously adding confining liquid, ultrasonically mixing uniformly and centrifuging; collecting the precipitate, placing the precipitate in a glycine solution, and performing ultrasonic dispersion to obtain a compound of the fluorescent microspheres and the antibody; the coupling method can ensure the full contact of each reactant, can obviously improve the coupling efficiency and the coupling strength of the fluorescent microspheres and the antibody, ensures that the surfaces of the microspheres are fully combined with the binding sites of antibody protein, can effectively improve the stability of the prepared compound, and avoids the occurrence of agglutination phenomenon; in addition, the sensitivity of fluorescence detection can be improved.
Description
Technical Field
The embodiment of the invention relates to the technical field of biological analysis, in particular to a coupling method of fluorescent microspheres and antibodies.
Background
The detection of certain antigens or specific proteins is generally applied to immunolabeling techniques. The immunolabeling technology is a technology in which a substance which is easy to measure and has high sensitivity is labeled on a specific antigen or antibody protein, and the nature and the content of the antigen or antibody in a reaction system are displayed by the enhanced amplification of the label.
The markers commonly used at present comprise colloidal gold, latex, fluorescein, fluorescent microspheres, enzyme, radionuclide and the like. However, in the existing process of labeling the antibody by the fluorescent microsphere, the coupling efficiency and coupling strength of the fluorescent microsphere and the antibody are low due to unreasonable labeling methods, so that the fluorescence intensity is low.
Disclosure of Invention
Therefore, the embodiment of the invention provides a coupling method of a fluorescent microsphere and an antibody, so as to solve the problem that the coupling efficiency and coupling intensity of the fluorescent microsphere and the antibody are low and the fluorescence intensity is low due to unreasonable labeling methods in the prior art.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:
according to a first aspect of the embodiments of the present invention, there is provided a coupling method of a fluorescent microsphere and an antibody, the coupling method comprising the steps of:
(a) activating the fluorescent microspheres to obtain activated fluorescent microspheres;
(b) adding MES buffer solution containing glycerol into the activated fluorescent microspheres, ultrasonically mixing, then adding antibody solution, ultrasonically reacting, and rotationally mixing in a dark place;
(c) after the reaction is finished, adding confining liquid into the precipitate, ultrasonically mixing uniformly, rotating and mixing uniformly in a dark place, centrifuging, removing supernatant, and then continuously adding the confining liquid, ultrasonically mixing uniformly and centrifuging;
(d) and collecting the centrifuged precipitate, placing the precipitate in a glycine solution, and performing ultrasonic dispersion to obtain the compound of the fluorescent microspheres and the antibody.
According to the coupling method, the fluorescent microspheres are subjected to activation treatment, and a specific buffer solution and a uniform mixing mode are selected, so that the full contact of reactants is guaranteed, the coupling efficiency and the coupling strength of the fluorescent microspheres and the antibody can be remarkably improved, the surfaces of the microspheres are fully combined with the binding sites of the antibody protein, the stability of a prepared compound can be effectively improved, and the occurrence of an agglutination phenomenon is avoided; in addition, the sensitivity of fluorescence detection can be improved.
Further, in the step (b), the ultrasonic reaction time is 8-12 min.
Further, the surface of the fluorescent microsphere contains carboxyl, and the fluorescent microsphere is selected from any one of red fluorescent microsphere, green fluorescent microsphere, quantum dot fluorescent microsphere and time-resolved fluorescent microsphere.
Preferably, the fluorescent microspheres have a particle size of 80-500 nm.
Further, the antibody is a myoglobin monoclonal antibody.
According to the invention, through the limitation on the types and particle sizes of the fluorescent microspheres and the antibody protein, the coupling efficiency and coupling strength of the fluorescent microspheres and the antibody protein can be better improved, and the detection effect of immunofluorescence is improved.
In the invention, the ultrasonic blending, the lucifugal rotary blending and the centrifugation are not strictly limited, preferably, the ultrasonic frequency adopted by the ultrasonic blending is 40KHz, and the time is not less than 2 min;
the rotation speed of the light-resistant rotary mixing is 25-35r/min, and the time is 30-120 min; more preferably, in the step (b), the light-shielding rotation mixing time is 100-120 min; in the step (c), the mixture is rotated and mixed uniformly in a dark place for 30-50 min;
the centrifugal rotation speed is 12000-16000rpm, the centrifugal time is 15-25mnin, and the temperature is 3-5 ℃.
Further, the activation treatment comprises the following steps:
(1) adding MES buffer solution into the fluorescent microspheres, ultrasonically mixing uniformly, centrifuging, removing supernatant, continuously adding MES buffer solution, ultrasonically mixing uniformly, centrifuging, and collecting precipitates;
(2) adding MES buffer solution into the precipitate, ultrasonically mixing the mixture, adding ethanol solution containing NHS and ethanol solution containing EDC into the uniformly mixed mixture, ultrasonically reacting for 5min, rotatably mixing the mixture in a dark place, centrifuging the mixture to remove supernatant, adding citric acid buffer solution, ultrasonically mixing the mixture and centrifuging the mixture to obtain the activated fluorescent microspheres.
Furthermore, the volume ratio of the addition amount of the MES buffer solution to the fluorescent microspheres is (8-10) to 1; the concentration of the MES buffer is 48-52 mM.
Further, the concentration of NHS in the ethanol solution containing NHS is 8-12 mg/ml; the concentration of EDC in the ethanol solution containing EDC is 8-12 mg/ml.
Further, in the step (2), the volume ratio of the addition amount of the citric acid buffer solution to the fluorescent microspheres is 10: 1; preferably, the citric acid buffer is 0.01M citric acid buffer at pH 6.5.
Further, in the step (2), the rotation speed of the light-shielding rotary blending is 25-35r/min, and the time is 30-50 min.
The invention can fully improve the activity of the fluorescent microsphere and improve the coupling strength and coupling efficiency of the fluorescent microsphere and the antibody by the specific activation method.
Furthermore, the volume ratio of the addition amount of the MES buffer solution containing glycerol to the fluorescent microspheres is (9-11) to 1; the volume ratio of the addition amount of the antibody solution to the fluorescent microspheres is 1: 4-6, and the concentration of the antibody in the antibody solution is 0.8-3 mg/ml.
According to the invention, by controlling the addition amount of the antibody and the fluorescent microsphere, the antibody and the fluorescent microsphere can be fully combined, and have better space conformation, so that the reduction of the activity of antibody protein is avoided, and the detection effect of fluorescence detection is not influenced.
Further, in the step (c), the blocking solution is 20% BSA, and the BSA concentration in the reaction solution after the addition is 0.4-0.6%; the sealing time is 55-65 min.
Further, in the step (d), the concentration of glycine in the glycine solution is 4.5-5.5%.
The embodiment of the invention has the following advantages:
(1) according to the coupling method, the fluorescent microspheres are subjected to activation treatment, and a specific buffer solution and a uniform mixing mode are selected, so that the full contact of reactants is guaranteed, the coupling efficiency and the coupling strength of the fluorescent microspheres and the antibody can be remarkably improved, the surfaces of the microspheres are fully combined with the binding sites of the antibody protein, the stability of a prepared compound can be effectively improved, and the occurrence of an agglutination phenomenon is avoided; in addition, the sensitivity of fluorescence detection can be improved.
(2) According to the invention, through the limitation on the types and particle sizes of the fluorescent microspheres and the antibody protein, the coupling efficiency and coupling strength of the fluorescent microspheres and the antibody protein can be better improved, and the detection effect of immunofluorescence is improved.
(3) According to the invention, by controlling the addition amount of the antibody and the fluorescent microsphere, the antibody and the fluorescent microsphere can be fully combined, and have better space conformation, so that the reduction of the activity of antibody protein is avoided, and the sensitivity of fluorescence detection is influenced.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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.
Example 1
The embodiment is a coupling method of fluorescent microspheres and antibodies, which comprises the following steps:
(a) adding 800 mu L of 52mM MES buffer solution into 100 mu L of fluorescent microspheres, ultrasonically mixing, centrifuging, discarding supernatant, continuously adding 800 mu L of 52mM MES buffer solution, ultrasonically mixing, centrifuging, and collecting precipitate; adding 800 mu L of 52mM MES buffer solution into the precipitate, ultrasonically mixing the mixture, adding ethanol solution containing NHS and ethanol solution containing EDC into the uniformly mixed mixture, ultrasonically reacting the mixture for 5min, uniformly mixing the mixture in a dark rotating way, centrifuging the mixture to remove supernatant, then adding 1000 mu L of citric acid buffer with pH6.5 and 0.01M, ultrasonically mixing the mixture, and centrifuging the mixture to obtain the activated fluorescent microsphere;
wherein the concentration of NHS in the ethanol solution containing NHS is 8-12 mg/ml; the concentration of EDC in the ethanol solution containing EDC is 8-12 mg/ml; ultrasonic mixing is carried out by adopting 40KHz ultrasonic wave for 2 min; centrifugation is 15mnin at 16000rpm at 3 ℃; rotating in dark place, mixing at 25r/min for 50 min;
(b) adding 900 μ L MES buffer solution containing 0.5% glycerol into the activated fluorescent microsphere, treating with 40KHz ultrasonic for 2min, adding 17 μ L antibody solution 3mg/ml, treating with 40KHz ultrasonic for 10min, and rotating and mixing at 35r/min in dark for 100 min;
(c) after the reaction is finished, adding 20% BSA into the reaction solution until the BSA concentration in the reaction solution is 0.4%, carrying out blocking reaction for 65min, and centrifuging at 10000r/min for 10 min;
(d) and collecting the centrifuged precipitate, putting the precipitate into a glycine solution with the concentration of 4.5%, and performing ultrasonic dispersion to obtain the compound of the fluorescent microspheres and the antibody coupling.
Example 2
The embodiment is a coupling method of fluorescent microspheres and antibodies, which comprises the following steps:
(a) adding 1000 μ L of 48mM MES buffer solution into 100 μ L of fluorescent microsphere, ultrasonically mixing, centrifuging, discarding supernatant, continuously adding 1000 μ L of 48mM MES buffer solution, ultrasonically mixing, centrifuging, and collecting precipitate; adding 1000 mu L of 48mM MES buffer solution into the precipitate, ultrasonically mixing the mixture, adding ethanol solution containing NHS and ethanol solution containing EDC into the uniformly mixed mixture, ultrasonically reacting the mixture for 5min, uniformly mixing the mixture in a dark rotating way, centrifuging the mixture to remove supernatant, then adding 1000 mu L of citric acid with pH6.5 and 0.01M, ultrasonically mixing the mixture, and centrifuging the mixture to obtain the activated fluorescent microsphere;
wherein the concentration of NHS in the ethanol solution containing NHS is 8-12 mg/ml; the concentration of EDC in the ethanol solution containing EDC is 8-12 mg/ml; ultrasonic mixing is carried out by adopting 40KHz ultrasonic wave for 3 min; centrifugation is 25mnin at 12000rpm at 5 ℃; rotating and uniformly mixing in a dark place at the rotating speed of 35r/min for 30 min;
(b) adding 1100 μ L MES buffer solution containing 0.5% glycerol into the activated fluorescent microsphere, treating with 40KHz ultrasonic wave for 3min, adding 25 μ L0.8mg/ml antibody solution, treating with 40KHz ultrasonic wave for 10min, and rotating and mixing at 25r/min for 120min in dark;
(c) after the reaction is finished, adding 20% BSA into the reaction solution until the BSA concentration in the reaction solution is 0.4%, carrying out blocking reaction for 65min, and centrifuging at 10000r/min for 10 min;
(d) and collecting the centrifuged precipitate, putting the precipitate into a glycine solution with the concentration of 4.5%, and performing ultrasonic dispersion to obtain the compound of the fluorescent microspheres and the antibody coupling.
Example 3
The embodiment is a coupling method of fluorescent microspheres and antibodies, which comprises the following steps:
(a) adding 900 mu L of 50mM MES buffer solution into 100 mu L of fluorescent microspheres, ultrasonically mixing, centrifuging, discarding supernatant, continuously adding 1000 mu L of 50mM MES buffer solution, ultrasonically mixing, centrifuging, and collecting precipitate; adding 975 mu L of 50mM MES buffer solution into the precipitate, ultrasonically mixing the mixture, adding ethanol solution containing NHS and ethanol solution containing EDC into the uniformly mixed mixture, ultrasonically reacting the mixture for 5min, uniformly mixing the mixture in a dark rotating way, centrifuging the mixture to remove supernatant, then adding 1000 mu L of citric acid with pH6.5 and 0.01M for buffering, ultrasonically mixing the mixture and centrifuging the mixture to obtain the activated fluorescent microsphere;
wherein the concentration of NHS in the ethanol solution containing NHS is 8-12 mg/ml; the concentration of EDC in the ethanol solution containing EDC is 8-12 mg/ml; ultrasonic mixing by adopting … ultrasonic wave for 2 min; centrifugation is 20mnin at 14000rpm at 4 ℃; rotating in dark place, mixing uniformly at a rotation speed of 30r/min for 40 min;
(b) adding 980 mu L of MES buffer solution containing 0.5% of glycerol into the activated fluorescent microspheres, processing for 2min by adopting 40KHz ultrasonic waves, then adding 20 mu L of 1.0mg/ml antibody solution, then processing for 2min by adopting 40KHz ultrasonic waves, and then uniformly mixing for 120min in a dark rotating way at 30 r/min;
(c) after the reaction is finished, adding 20% BSA into the reaction solution until the BSA concentration in the reaction solution is 0.4%, carrying out blocking reaction for 65min, and centrifuging at 10000r/min for 10 min;
(d) and collecting the centrifuged precipitate, putting the precipitate into a glycine solution with the concentration of 4.5%, and performing ultrasonic dispersion to obtain the compound of the fluorescent microspheres and the antibody coupling.
Comparative example 1
The comparison example is a coupling method of a conventional fluorescent microsphere and an antibody, and the coupling method is basically the same as the coupling method in the example 1, except that the fluorescent microsphere is diluted by adopting an MES buffer solution before the antibody is added in the step (b), and after the antibody is added, the reaction is carried out for 10min under the condition of ultrasonic treatment.
Experimental example 1
The immunofluorescence test paper is prepared by adopting the fluorescent microsphere coupling antibody prepared in the embodiment 1, and the preparation method comprises the following steps:
(a) preparation of marking pad: fluorescent microsphere-conjugated antibody was prepared according to the procedure of example 1, diluted to 5% with PBS solution (pH) containing 1% BSA, 0.1% Triton, 10% sucrose, 0.2% PVP40, and sprayed on a label pad treated with the same solution using a three-dimensional film-scribing metal sprayer at a spray rate of 4. mu.L/cm; then placing the sprayed cushion in an oven at 37 ℃, drying for 4 hours, and then placing under the condition of humidity of less than 20% for hermetic storage;
(b) preparation of coating film: respectively diluting the myoglobin monoclonal antibody and Protein A to 0.5mg/mL and 0.2mg/mL by using a membrane-cutting diluent, and respectively spraying the diluted solutions onto a nitrocellulose membrane to serve as a detection line (T) and a quality control line (C), wherein the coating amount is 1 mu L/cm, and the interval between the detection line and the quality control line is 4 mm; then placing the mixture in a 37 ℃ oven, drying the mixture for 12 hours, and then placing the mixture under the condition of humidity of less than 20% for sealed storage; the scribing diluent is a PBS solution (pH7.4) containing 2% of sucrose;
(c) assembling the detection card: and sequentially adhering the water absorption pad, the coating film, the marking pad, the sample pad and the identification strip on a PVC (polyvinyl chloride) base plate, cutting the PVC base plate into test strips with the width of 4mm, and filling the test strips into a card shell to obtain the immunofluorescence detection card for detecting the myoglobin.
The immunofluorescence test paper is prepared by the method by coupling the fluorescent microspheres with the antibody in the comparative example 1;
the test paper prepared by the fluorescent microsphere antibody conjugates prepared by the two different coupling methods of example 1 and comparative example 1 is respectively detected, and the chromogenic fluorescence intensity of the two conjugates is compared.
The detection method comprises the following steps:
preparing myoglobin antigen into 0ng/mL, 58ng/mL and 580ng/mL by PBS, respectively using two detection cards, testing each concentration point for 10 times, and comparing fluorescence intensity values of a detection line (T) and a quality control line (C); the experimental results are as follows:
table 1 test paper strip test results prepared by using fluorescent microsphere coupled antibody in example 1
Table 2 test paper strip test results prepared by using fluorescent microsphere-conjugated antibody in comparative example 1
As can be seen from tables 1 and 2:
compared with the coupling method in the comparative example 1, the coupling method in the embodiment of the application has the advantages that the compound obtained by coupling has higher activity, the coupling efficiency and the coupling strength of the fluorescent microspheres and the antibody protein can be better improved, and the immunofluorescence detection effect is improved.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A coupling method of fluorescent microspheres and antibodies is characterized by comprising the following steps:
(a) activating the fluorescent microspheres to obtain activated fluorescent microspheres;
(b) adding MES buffer solution containing glycerol into the activated fluorescent microspheres, ultrasonically mixing, then adding antibody solution, ultrasonically reacting, and rotationally mixing in a dark place;
(c) after the reaction is finished, adding confining liquid into the precipitate, ultrasonically mixing uniformly, rotating and mixing uniformly in a dark place, centrifuging, removing supernatant, and then continuously adding the confining liquid, ultrasonically mixing uniformly and centrifuging;
(d) and collecting the centrifuged precipitate, placing the precipitate in a glycine solution, and performing ultrasonic dispersion to obtain the compound of the fluorescent microspheres and the antibody.
2. The coupling process according to claim 1, characterized in that said activation treatment comprises the following steps:
(1) adding MES buffer solution into the fluorescent microspheres, ultrasonically mixing uniformly, centrifuging, removing supernatant, continuously adding MES buffer solution, ultrasonically mixing uniformly, centrifuging, and collecting precipitates;
(2) adding MES buffer solution into the precipitate, ultrasonically mixing the mixture, adding ethanol solution containing NHS and ethanol solution containing EDC into the uniformly mixed mixture, ultrasonically reacting for 5min, rotatably mixing the mixture in a dark place, centrifuging the mixture to remove supernatant, adding citric acid buffer solution, ultrasonically mixing the mixture and centrifuging the mixture to obtain the activated fluorescent microspheres.
3. The coupling method of claim 2, wherein the MES buffer is added in an amount such that the volume ratio of the MES buffer to the fluorescent microspheres is (8-10) to 1; the concentration of the MES buffer is 48-52 mM.
4. The coupling method according to claim 2, wherein the concentration of NHS in the ethanol solution containing NHS is 8-12 mg/ml; the concentration of EDC in the ethanol solution containing EDC is 8-12 mg/ml.
5. The coupling method according to claim 1, wherein the surface of the fluorescent microsphere contains carboxyl, and the fluorescent microsphere is selected from any one of red fluorescent microsphere, green fluorescent microsphere, quantum dot fluorescent microsphere and time-resolved fluorescent microsphere.
6. The coupling method according to claim 1, wherein the fluorescent microspheres have a particle size of 80-500 nm.
7. The method of coupling of claim 1, wherein the antibody is a myoglobin monoclonal antibody.
8. The coupling method according to claim 1, wherein the MES buffer containing glycerol is added in an amount to provide a volume ratio of fluorescent microspheres of (9-11) to 1; the volume ratio of the addition amount of the antibody solution to the fluorescent microspheres is 1: 4-6, and the concentration of the antibody in the antibody solution is 0.8-3 mg/ml.
9. The coupling method according to claim 1, wherein in the step (c), the blocking solution is 20% BSA, and the concentration of BSA in the reaction solution after the addition is 0.4-0.6%; the sealing time is 55-65 min.
10. The coupling process of claim 1, wherein in step (d) the glycine concentration in the glycine solution is from 4.5 to 5.5%.
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| CN111551730A (en) * | 2020-05-18 | 2020-08-18 | 上海艾瑞德生物科技有限公司 | Fluorescent microsphere sealing liquid and kit using same |
| CN111879921A (en) * | 2020-06-22 | 2020-11-03 | 武汉生之源生物科技股份有限公司 | Fluorescent microsphere of coupled antibody and preparation method and application thereof |
| CN114113578A (en) * | 2021-11-30 | 2022-03-01 | 湖南永和阳光生物科技股份有限公司 | Method for coupling fluorescent microspheres with antibody and application thereof |
| CN115656494A (en) * | 2022-10-17 | 2023-01-31 | 济南诺磐生物科技有限公司 | Method for coupling blue microspheres with antibody and application |
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