CN112649599B - Method for indirectly marking and sealing colloidal gold - Google Patents
Method for indirectly marking and sealing colloidal gold Download PDFInfo
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- CN112649599B CN112649599B CN202011504889.9A CN202011504889A CN112649599B CN 112649599 B CN112649599 B CN 112649599B CN 202011504889 A CN202011504889 A CN 202011504889A CN 112649599 B CN112649599 B CN 112649599B
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
Abstract
The invention relates to the technical field of biological analysis and detection, in particular to a method for indirectly labeling and sealing colloidal gold. According to the method, the stability of the protein conjugate on the surface of the colloidal gold is obviously enhanced by utilizing the macromolecule blocking agent for blocking and then utilizing the micromolecule blocking agent for blocking, and the coupling efficiency, sensitivity and stability can be effectively improved by utilizing the protein conjugate for immunoassay.
Description
Technical Field
The invention relates to the technical field of biological analysis and detection, in particular to a method for indirectly labeling and sealing colloidal gold.
Background
Colloidal gold labeling is essentially a coating process in which a polymer such as a protein is directly adsorbed onto the surface of a colloidal gold particle. The adsorption mechanism is that macromolecules such as protein and the like form a relatively stable compound through interaction between one or more acting forces and colloidal metallography, and the acting forces are electrostatic action, hydrophobic action, gold-sulfhydryl covalent bonding and the like. After the interaction between the colloidal gold and the macromolecular substance, usually blocking with a non-target protein such as BSA, due to steric hindrance, there are many blank sites on the surface of the colloidal gold, which will adsorb proteins and specific small molecular substances, and compete with the adsorbed target protein, resulting in the target protein falling off and poor stability. The colloidal gold product prepared by the compound of the colloidal gold and the target protein has the advantages of convenience, rapidness, no need of instruments and the like, and is widely applied to the field of biological detection.
The chinese patent 'a colloidal gold labeling method' with application number 201110044743.5 describes that protein is directly labeled on colloidal gold particles by changing the pH of the labeling buffer, which improves stability and sensitivity. The chinese patent application No. 201310485455.2, "a covalent labeling method of colloidal gold for rapid detection", describes that protein is treated to introduce more thiol groups, and covalent bonds are formed through gold-thiol groups, so that protein is covalently coupled on the surface of gold particles. In these 2 patents, the method of gold-conjugated protein has a common feature that the target protein and gold particles are directly contacted and adsorbed onto the surface of gold particles, the binding is a random adsorption process, the spatial distance between the protein and gold particles is very small, and it is very easy to shield the reaction sites in the random adsorption process, resulting in reduced reactivity of the labeled protein and low labeling efficiency.
The chinese patent No. 201810498754.2, "a method for preparing a gold-labeled immunochromatographic test strip in which colloidal gold is labeled with a goat-anti-mouse secondary antibody and then indirectly linked with a mouse-anti label," describes a method for indirectly coupling a secondary antibody and a monoclonal antibody on the surface of colloidal gold. Chinese patent No. 201811168958.6, "antibody detection capture method for indirectly labeling nanoparticles and kit thereof" describes that a labeled antibody is adsorbed on the surface of colloidal gold, followed by labeled protein coupling. The two patents are the indirect labeling protein on the surface of the colloidal gold, the common property of the two is that the indirect coupling protein on the surface of the colloidal gold improves the reactivity and the specificity, the coupling is non-specific physical adsorption in the first stage, the coupling is indirect coupling target protein by utilizing the specific reaction in the second stage, after the first step of adsorption, because a plurality of blank sites are arranged on the surface of the colloidal gold, the sites can adsorb non-labeling substances and compete with the adsorbed target protein, the target protein is caused to fall off, and the stability is poor.
The Chinese patent 'detection marker of specific antibody, preparation method, application and kit' with application number 202010403046.3 describes that glycine is used as a blocking agent after colloidal gold is coupled with macromolecules. The Chinese patent 'immune colloidal gold test paper and its method for quantitatively detecting CLB by matching with photoelectric sensor' with application No. 201210027887.4 describes the use of mixture of skimmed milk powder and glycine as blocking agent. The blocking effect of the blocking agents is not ideal, so that the problems of low coupling efficiency and poor stability of immunoassay are caused.
Disclosure of Invention
In view of the above, the present invention provides a method for indirectly labeling and sealing with colloidal gold. The method combines a sealing method of utilizing a micromolecular sealing agent after sealing by utilizing a macromolecule sealing agent with the indirect labeling of colloidal gold, so that the stability of the protein conjugate on the surface of the colloidal gold is obviously enhanced, and the coupling efficiency, the sensitivity and the stability can be effectively improved by utilizing the protein conjugate to carry out immunoassay.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for indirectly marking and sealing colloidal gold, which comprises the following steps:
step 1: obtaining colloidal gold particles coated with a first conjugate, and then adding a first confining liquid for confinement to obtain a compound 1;
step 2: adding a second sealing liquid into the colloidal gold-first conjugate-first sealing agent compound for sealing to obtain a compound 2;
and step 3: adding a second conjugate into the complex 2 to obtain a colloidal gold protein conjugate;
the first blocking agent liquid consists of a first blocking agent and a buffer liquid, and the first blocking agent is selected from one or more of BSA, Casein, cerulenin, high PEG20000 and PEG 6000;
the second blocking agent liquid consists of a second blocking agent and a buffer liquid, and the second blocking agent is selected from one or more of glycine, arginine, lysine and histidine.
In some embodiments, the first blocking agent is BSA; the second sealant is glycine.
In some embodiments, the first sealant in the first sealant solution is 0.1-2% by mass; the mass percentage of the second sealant in the second sealant liquid is 0.1-2%.
In some embodiments, the buffer in the first and second blocking solutions is selected from one or more of PBS, Tris-HCl, CB, HEPES. Further, the buffer pH was 7.5.
In some embodiments, the first blocking solution is PBS buffer containing 0.1-2wt% BSA and the second blocking solution is PBS buffer containing 0.1-2wt% glycine. In some embodiments, the first blocking solution is PBS buffer containing 0.1 wt% BSA, and the second blocking solution is PBS buffer containing 0.1 wt% glycine. In some embodiments, the first blocking solution is PBS buffer containing 2wt% BSA, and the second blocking solution is PBS buffer containing 2wt% glycine.
In some embodiments, the first blocking solution is a PBS buffer comprising 0.1-2wt% PEG, and the second blocking solution is a PBS buffer comprising 0.1-2wt% histidine. In some embodiments, the first blocking solution is 0.1 wt% PEG6000 in PBS buffer, and the second blocking solution is 0.1 wt% histidine in PBS buffer.
In some embodiments, the first blocking solution is PBS buffer comprising 0.1-2wt% casein and the second blocking solution is PBS buffer comprising 0.1-2wt% arginine. In some embodiments, the first blocking solution is a PBS buffer solution containing 0.1 wt% casein, and the second blocking solution is a PBS buffer solution containing 0.1 wt% arginine
In the present invention, the first conjugate is one selected from the group consisting of a his-tag antibody, a Flag-tag antibody, a C-Myc-tag antibody, an HA-tag antibody and a GST-tag antibody. The second conjugate is a test protein labeled with a label, and the second conjugate is combined with the first conjugate (namely, a label antibody) through the label of the second conjugate. Wherein, the second conjugate is marked with a label to be detected protein, and the label is selected from one of a his label, a Flag label, a C-Myc label, an HA label and a GST label.
Furthermore, the protein to be detected comprises HBsAb, MP-IgM, MP-IgG, CP-IgM, COXB-IgM, RSV-IgM, ADV-IgM, SARS-CoV-2-Ab, SARS-CoV-2-IgM, SARS-CoV-2-IgG and the like. In some embodiments, the protein to be tested is SARS-CoV-2-IgG.
According to the method for indirectly labeling and sealing the colloidal gold, provided by the invention, the biological label and the labeled antibody are utilized to react with each other, the labeled antibody is adsorbed on the surface of the colloidal gold particles, and then the protein with the corresponding label reacts with the labeled antibody, so that a colloidal gold-labeled antibody-labeled protein compound is indirectly formed on the colloidal gold, on one hand, regular protein distribution is formed, on the other hand, due to the spatial distance, the sites are more exposed, and the reactivity and the labeling efficiency are greatly improved. And the stability of the protein conjugate on the surface of the colloidal gold is obviously enhanced by utilizing the macromolecule sealing agent for sealing and then utilizing the micromolecule sealing agent for sealing. Experimental results show that the protein conjugate can be used for immunoassay to effectively improve the coupling efficiency, sensitivity and stability.
Detailed Description
The invention provides a method for indirectly marking and sealing colloidal gold. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications in the methods and applications disclosed herein, or appropriate variations and combinations thereof, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 Indirect labeling and blocking method with colloidal gold according to the present invention (colloidal gold coupled SARS-CoV-2 recombinant N protein-his tag)
(1) Preparing colloidal gold: colloidal gold having an average particle diameter of about 40nm was prepared by reducing a 0.01% (mass unit) solution of trisodium citrate in a boiling solution of tetrachlorogold acid according to the fress method (fress, G., Preparation of gold dispersions varyingparticles size: controlled circulation for the regulation of particle size of gold in mineral centres in Nature: Physical Science241(1973), 20-22).
(2) Adjusting the pH value: 10ml of prepared colloidal gold is added with 0.2MK 2 CO 3 Adjusted to a pH of 7.5. + -. 0.05.
(3) Addition of the first conjugate: the first conjugate, murine anti-his-antibody, 20ug, was added in step 2 and reacted for 30 min.
(4) And (3) sealing: a first blocking solution (0.1 wt% BSA in PBS) was added to the reaction in step 3 and blocked for half an hour.
(5) Cleaning: and (4) centrifuging the reactant in the step 4 and removing the supernatant to obtain the colloidal gold-his antibody-BSA complex.
(6) And (3) sealing: a second blocking solution (0.1 wt% glycine in PBS buffer) was added at step 5 and blocking was added for 0.5 hours.
(7) Cleaning: centrifuging the reactant in the step 6 and removing the supernatant to obtain the colloidal gold-his antibody-BSA-glycine complex.
(8) Adding a second conjugate: colloidal gold conjugated SARS-CoV-2 recombinant N protein (his tag) 20 ug: adding into the compound of step 7.
(9) Coupling: in step 8, 10ml of reaction buffer 0.05MPB pH8.0 (containing 2mg/ml BSA and 1mg/ml sucrose) was added, and the reaction was carried out at 20 to 25 ℃ for 60 minutes with shaking.
(10) Cleaning: centrifuging the reactant in the step 9 and removing the supernatant to obtain the colloidal gold-target protein compound. Stored in 3ml0.05M PB pH7.4 (containing 5mg/ml BSA and 1mg/ml arginine). And 4 ℃ storing for later use.
Example 2
The invention relates to an indirect marking and sealing method (colloidal gold coupling SARS-CoV-2 recombination N protein-his label)
(1) Preparing colloidal gold: colloidal gold having an average particle diameter of about 40nm was prepared by reducing a 0.01% (mass unit) solution of trisodium citrate in a boiling solution of tetrachlorogold acid according to the fress method (fress, G., Preparation of gold dispersions varyingparticles size: controlled circulation for the regulation of particle size of gold in mineral centres in Nature: Physical Science241(1973), 20-22).
(2) Adjusting the pH value: 10ml of the prepared gold colloid was adjusted to pH 7.5. + -. 0.05 using 0.2M K2CO 3.
(3) Addition of the first conjugate: the first conjugate, murine anti-his-antibody, 20ug, was added in step 2 and reacted for 30 min.
(4) And (3) sealing: a first blocking solution (0.1 wt% PEG6000 in PBS buffer) was added to the reaction in step 3 and blocked for half an hour.
(5) Cleaning: and (4) centrifuging the reactant in the step (4) and removing the supernatant to obtain the colloidal gold-his antibody-PEG 6000 compound.
(6) And (3) sealing: a second blocking solution (0.1 wt% histidine in PBS buffer) was added at step 5, and blocking was added for 0.5 hours.
(7) Cleaning: centrifuging the reactant in the step 6 and removing the supernatant to obtain the colloidal gold-his antibody-PEG 6000-histidine compound.
(8) Adding a second conjugate: colloidal gold conjugated SARS-CoV-2 recombinant N protein (his tag) 20 ug: adding into the compound of step 7.
(9) Coupling: in step 8, 10ml of reaction buffer 0.05MPB pH8.0 (containing 2mg/ml BSA and 1mg/ml sucrose) was added, and the reaction was carried out at 20 to 25 ℃ for 60 minutes with shaking.
(10) Cleaning: centrifuging the reactant in the step 9 and removing the supernatant to obtain the colloidal gold-target protein compound. Stored in 3ml0.05M PB pH7.4 (containing 5mg/ml BSA and 1mg/ml arginine). And 4 degrees are stored for later use.
Example 3
The invention relates to an indirect marking and sealing method (colloidal gold coupling SARS-CoV-2 recombination N protein-his label)
(1) Preparing colloidal gold: colloidal gold having an average particle diameter of about 40nm was prepared by reducing a 0.01% (mass unit) solution of trisodium citrate in a boiling solution of tetrachlorogold acid according to the fress method (fress, G., Preparation of gold dispersions varyingparticles size: controlled circulation for the regulation of particle size of gold in mineral centres in Nature: Physical Science241(1973), 20-22).
(2) Adjusting the pH value: 10ml of the prepared gold colloid was adjusted to pH 7.5. + -. 0.05 using 0.2M K2CO 3.
(3) Addition of the first conjugate: the first conjugate, murine anti-his-antibody, 20ug, was added in step 2 and reacted for 30 min.
(4) And (3) sealing: a first blocking solution (0.1 wt% casein PBS buffer) was added to the reaction in step 3 and blocked for half an hour.
(5) Cleaning: and (4) centrifuging the reactant in the step 4 and removing the supernatant to obtain the colloidal gold-his antibody-casein compound.
(6) And (3) sealing: a second blocking solution (0.1 wt% arginine in PBS buffer) was added at step 5, and blocking was added for 0.5 hours.
(7) Cleaning: and (4) centrifuging the reactant in the step 6 and removing the supernatant to obtain the colloidal gold-his antibody-casein-arginine complex.
(8) Adding a second conjugate: colloidal gold conjugated SARS-CoV-2 recombinant N protein (his tag) 20 ug: adding into the compound of step 7.
(9) Coupling: in step 8, 10ml of reaction buffer 0.05MPB pH8.0 (containing 2mg/ml BSA and 1mg/ml sucrose) was added, and the reaction was carried out at 20 to 25 ℃ for 60 minutes with shaking.
(10) Cleaning: centrifuging the reactant in the step 9 and removing the supernatant to obtain the colloidal gold-target protein compound. Stored in 3ml0.05M PB pH7.4 (containing 5mg/ml BSA and 1mg/ml arginine). And 4 degrees are stored for later use.
Comparative example 1 Indirect labeling, macromolecule blocking method (colloidal gold coupling SARS-CoV-2 recombinant N protein-his tag)
(1) Preparing colloidal gold: colloidal gold having an average particle diameter of about 40nm was prepared by reducing a 0.01% (mass unit) solution of trisodium citrate in a boiling solution of tetrachlorogold acid according to the fress method (fress, G., Preparation of gold dispersions varyingparticles size: controlled circulation for the regulation of particle size of gold in mineral centres in Nature: Physical Science241(1973), 20-22).
(2) Adjusting the pH value: 10ml of prepared colloidal gold is added with 0.2MK 2 CO 3 Adjusted to a pH of 7.5. + -. 0.05.
(3) Addition of the first conjugate: the first conjugate, murine anti-his-antibody, 20ug, was added in step 2 and reacted for 30 min.
(4) And (3) sealing: to the reaction of step 3, 0.1g BSA was added and blocked for half an hour.
(5) Cleaning: and (4) centrifuging the reactant in the step 4 and removing the supernatant to obtain the colloidal gold-his antibody-BSA complex.
(6) Adding a second conjugate: colloidal gold conjugated SARS-CoV-2 recombinant N protein (his tag) 20 ug: adding into the compound of step 5.
(7) Coupling: in step 6, 10ml of reaction buffer 0.05MPB pH8.0 (containing 2mg/ml BSA and 1mg/ml sucrose) was added, and the reaction was carried out at 20 to 25 ℃ for 60 minutes with shaking.
(8) Cleaning: centrifuging the reactant in the step 9 and removing the supernatant to obtain the colloidal gold-target protein compound. Stored in 3ml0.05M PB pH7.4 (containing 5mg/ml BSA and 1mg/ml arginine). And 4 ℃ storing for later use.
Comparative example 2 Indirect labeling, Large and Small molecule Mixed blocking method (colloidal gold coupled SARS-CoV-2 recombinant N protein-his tag)
(1) Preparing colloidal gold: colloidal gold having an average particle diameter of about 40nm was prepared by reducing a 0.01% (mass unit) solution of trisodium citrate in a boiling solution of tetrachlorogold acid according to the fress method (fress, G., Preparation of gold dispersions varyingparticles size: controlled circulation for the regulation of particle size of gold in mineral centres in Nature: Physical Science241(1973), 20-22).
(2) Adjusting the pH value: 10ml of the prepared gold colloid was adjusted to pH 7.5. + -. 0.05 using 0.2M K2CO 3.
(3) Addition of the first conjugate: the first conjugate, murine anti-his-antibody, 20ug, was added in step 2 and reacted for 30 min.
(4) And (3) sealing: blocking solution (PBS buffer containing 0.1 wt% BSA and 0.1 wt% glycine) was added to the reaction in step 3 and blocked for half an hour.
(5) Cleaning: and (4) centrifuging the reactant in the step 4 and removing the supernatant to obtain the colloidal gold-his antibody-BSA glycine complex.
(6) Adding a second conjugate: colloidal gold conjugated SARS-CoV-2 recombinant N protein (his tag) 20 ug: adding into the compound of step 5.
(7) Coupling: in step 6, 10ml of reaction buffer 0.05MPB pH8.0 (containing 2mg/ml BSA and 1mg/ml sucrose) was added, and the reaction was carried out at 20 to 25 ℃ for 60 minutes with shaking.
(8) Cleaning: centrifuging the reactant in the step 9 and removing the supernatant to obtain the colloidal gold-target protein compound. Stored in 3ml0.05M PB pH7.4 (containing 5mg/ml BSA and 1mg/ml arginine). And 4 degrees are stored for later use.
Comparative example 3 direct labeling, large and small molecule hybrid confinement method
(1) Preparing colloidal gold: colloidal gold having an average particle diameter of about 40nm was prepared by reducing a 0.01% (mass unit) solution of trisodium citrate in a boiling solution of tetrachlorogold acid according to the fress method (fress, G., Preparation of gold dispersions varying particle size: controlled circulation for the regulation of the particle size of the gold particles in the nature of Physical Science241(1973), 20-22).
(2) Adjusting the pH value: 10ml of prepared colloidal gold is added with 0.2MK 2 CO 3 Adjusted to a pH of 7.5. + -. 0.05.
(3) Adding a coupling protein: SARS-CoV-2 recombinant N protein (his tag) was added at 20ug in step 2 and reacted for 30 min.
(4) And (3) sealing: blocking solution (PBS buffer containing 0.1 wt% BSA and 0.1 wt% glycine) was added to the reaction in step 3 and blocked for half an hour.
(5) Cleaning: and (4) centrifuging the reactant in the step (4) and removing the supernatant to obtain the colloidal gold-protein conjugate. Stored in 3ml0.05M PB pH7.4 (containing 5mg/ml BSA and 1mg/ml arginine). And 4 degrees are stored for later use.
Comparative example 4 direct labeling and mixed sealing method for large and small molecules
(1) Preparing colloidal gold: colloidal gold having an average particle diameter of about 40nm was prepared by reducing a 0.01% (mass units) solution of trisodium citrate in a boiling solution of tetrachlorogold acid according to the fress method (fress, G., Preparation of gold dispersions varying particle size: controlled circulation for the regulation of particle size of gold particles in mineral sumptions in Nature: Physical Science241(1973), 20-22).
(2) Adjusting the pH value: 10ml of prepared colloidal gold is used, 0.2M K 2 CO 3 Adjusting to pH7.5. + -. 0.05 values.
(3) Adding a coupling protein: SARS-CoV-2 recombinant N protein (his tag) 160ug was added in step 2 and reacted for 30 min.
(4) And (3) sealing: blocking solution (PBS buffer containing 0.1 wt% BSA and 0.1 wt% glycine) was added to the reaction in step 3 and blocked for half an hour.
(5) Cleaning: and (4) centrifuging the reactant in the step (4) and removing the supernatant to obtain the colloidal gold-protein conjugate. Stored in 3ml0.05M PB pH7.4 (containing 5mg/ml BSA and 1mg/ml arginine). 4 degrees for standby.
Test examples
The five colloidal gold conjugates prepared in example 1 and comparative examples 1 to 4 were sprayed on 5 glass fiber pads at 5.0ul/cm and dried at 37 degrees, and anti-human IgG antibody was coated on NC membrane at 1.0mg/ml and 1.2ul/cm, and dried at 37 degrees for use. And placed under a 45 degree seal. Sequentially adhering a glass cellulose membrane, a glass cellulose membrane coated with a labeled product, a water-absorbing glass cellulose membrane and a nitrocellulose membrane coated with an antibody on a single-sided plastic plate, finally adhering water-absorbing paper with the length of 30 cm and the width of 2 cm, absorbing redundant solution after chromatography, and tightly connecting the membranes to each other to prepare an immunochromatography test paper large plate, longitudinally cutting the adhered plastic plate into immune test paper strips with the width of 4mm by using a slitter, adding 10ul of serum to be detected at one end of a sample pad of the immune test paper strips with the width of 4mm, dripping 2 drops of diluent, and observing the result after chromatography is carried out for 15 minutes. The results are shown in Table 1.
TABLE 1
TABLE 2
Note: + + + + indicates strong positive color development; developing at positive and middle conditions; + weakly positive, color development can be seen; the + -expression is not easy to be interpreted
The result shows that the sensitivity of the colloidal gold indirect labeling and sealing method to the immunodetection is improved by about 8 times compared with the traditional method (embodiment 1VS comparative example 4), and the effect is obvious. The stability examination result shows that the test paper prepared by the method can still accurately detect the low-concentration quality control sample (16 multiplied by dilution) after being placed at 45 ℃ for 28 days, and has better stability and higher detection rate compared with comparative examples 1-4.
The stability and sensitivity of the colloidal gold conjugates prepared in examples 2 to 3 were examined according to the methods of the test examples, and the results were consistent with those of example 1.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (5)
1. A method for indirectly labeling and sealing colloidal gold, which is characterized by comprising the following steps:
step 1: obtaining colloidal gold particles coated with a first conjugate, and then adding a first confining liquid for confinement to obtain a compound 1;
step 2: adding a second sealing liquid into the colloidal gold-first conjugate-first sealing agent compound for sealing to obtain a compound 2;
and step 3: adding a second conjugate into the complex 2 to obtain a colloidal gold protein conjugate;
the first sealant liquid consists of a first sealant and a buffer;
the second sealant liquid consists of a second sealant and a buffer;
the first sealant and the second sealant are BSA, glycine or
The first sealant and the second sealant are PEG6000 and histidine in sequence,
the first sealant and the second sealant are Casein and arginine in sequence;
the mass percentage of the first sealing agent in the first sealing liquid is 0.1-2%; the mass percentage of the second sealing agent in the second sealing liquid is 0.1-2%;
the first conjugate is selected from one of a his tag antibody, a Flag tag antibody, a C-Myc tag antibody, an HA tag antibody and a GST tag antibody;
the second conjugate is a protein to be detected marked by a label, the label of the second conjugate is combined with the first conjugate, and the label is selected from one of his label, Flag label, C-Myc label, HA label and GST label.
2. The method of claim 1, wherein the first blocking agent is BSA;
the second sealant is glycine.
3. The method according to claim 1 or 2, wherein the buffer in the first and second blocking solutions is selected from one or more of PBS, Tris-HCl, CB and HEPES.
4. The method according to claim 3, wherein the buffer in the first blocking solution and the second blocking solution has a pH of 7.5.
5. The method of claim 1, wherein the first blocking solution is PBS buffer containing 0.1-2wt% BSA, and the second blocking solution is PBS buffer containing 0.1-2wt% glycine.
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