CN116699126A - Blocking agent of antibody-coupled microsphere complex - Google Patents
Blocking agent of antibody-coupled microsphere complex Download PDFInfo
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- CN116699126A CN116699126A CN202310699946.0A CN202310699946A CN116699126A CN 116699126 A CN116699126 A CN 116699126A CN 202310699946 A CN202310699946 A CN 202310699946A CN 116699126 A CN116699126 A CN 116699126A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Food Science & Technology (AREA)
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- General Health & Medical Sciences (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a blocking agent of an antibody-coupled microsphere compound, which comprises the following blocking liquid raw materials in percentage by weight: 70% -90% of buffer solution, 10% -30% of stabilizer, 0.01% -5% of surfactant and 0.1% -0.5% of preservative. The invention provides a method for preparing an antibody coupled microsphere compound, which is applied to an antibody coupled microsphere compound, can reduce the nonspecific adsorption of an immune detection reagent, can improve the sensitivity, repeatability and stability of detection, and can improve the performance of the detection reagent, and an added stabilizer mainly serves as a blocking agent to play a role in stabilizing the conformation of the detection reagent, so that the trend of signal attenuation is slowed down; the PB buffer solution has good buffer effect and good salt balance capability, can play a role in regulating osmotic pressure, can maintain the activity of an antibody in the reagent in a short period, greatly improves the stability of the reagent, and can obtain good stability when the blocking agent is used for blocking an antibody coupling microsphere compound.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a blocking agent of an antibody-coupled microsphere compound.
Background
The affinity between the antigen and the antibody can make the antigen and the antibody specifically combine, and the basic principle of the immunodetection technology is to perform qualitative, positioning or quantitative detection on the to-be-detected object in the sample according to the phenomenon and character characteristics of the antigen and the antibody after combining to form an immune composite structure. In recent years, the advantages of high specificity, rapidness, easy standardized operation and the like are rapidly developed in the fields of biomedicine, food detection, environmental monitoring and the like. In the current mainstream immunodiagnosis technologies such as lateral immunochromatography, latex enhanced turbidimetry, chemiluminescence and the like, the microspheres have extremely wide application scenes. The microsphere used as a marker or a carrier in immune reaction is subjected to antibody coupling modification, and after antigen-antibody specific reaction, the to-be-detected object can be analyzed according to different methodologies, so that qualitative and quantitative detection is realized. In different immune detection methods, the antibody-coupled microsphere complex is generally considered as one of key factors which can influence the performances of detection sensitivity, repeatability, stability and the like, and the blocking of redundant reaction sites of the antibody-coupled microsphere complex is an important step for improving the performances of the microsphere complex and even the detection results, and the blocking of the redundant reaction sites of the antibody-coupled microsphere complex is an important factor which can influence the performances of the detection results at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the blocking agent of the antibody-coupled microsphere complex, which has the advantages of reducing non-specific interference in the immune reaction process and improving the sensitivity, repeatability and stability of the detection technology.
An antibody-coupled microsphere complex blocking agent comprises the following blocking liquid raw materials in percentage by weight: 70% -90% of buffer solution, 10% -30% of stabilizer, 0.01% -5% of surfactant and 0.1% -0.5% of preservative.
Preferably, the buffer solution is selected from one or more of PB buffer solution and Tris-hydrochloric acid; the PB buffer solution comprises purified water, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride, and the inventor finds that in the PB buffer solution, phosphate plays a role in buffering, so as to maintain a certain pH environment and regulate osmotic pressure, and the PB buffer solution mainly plays roles in maintaining stable pH, maintaining osmotic pressure and providing basic nutrition in a short time, can maintain the activity of bioactive products such as proteins, cells, tissues and organs in a short time, and provides necessary nutritional environment.
Preferably, the stabilizer is selected from bovine serum albumin, casein,And one or more of gels, wherein the blocking principle of bovine serum albumin is that the surface of a solid support has a plurality of holes, the protein on the gel is transferred to a membrane by electrotransformation, or the protein is bound to the surface by a mechanical filling or adsorption mode on an antigen/antibody fixing plate, the protein in a blocking solution can be bound with blank positions on the surface, and the protein can be bound to the membrane by a mechanical filling (stacking) and adsorption covering mode, and the blocking is said to be realized because a non-specific binding of primary antibodies is avoided by filling and covering protein binding sites. The bovine serum albumin can be combined with blank sites on the surface after the antibody is coupled with the microsphere compound, so that the nonspecific adsorption after the antibody is coupled with the latex microsphere is reduced, and the detection sensitivity of the reagent is improved; casein, which is a natural protein, has good biocompatibility and biodegradability, and thus is widely used in the fields of medicine and the like, and casein can block other substances to form a stable complex. However, the implementation of the casein blocking principle needs to meet certain conditions, such as proper ph, temperature, ionic strength and the like, under which casein can form a complex with other substances, so that the blocking effect is realized, the casein is not easy to dissolve, and the preparation is difficult, so that the casein is not the optimal choice of protein; /> Is an aqueous solution of high-purity pig dermis collagen polypeptide fragment, is easily dissolved in water, electrolyte solution and the like, and is always used for treating the skin diseasesThe additive for inert protein stabilizer, cell culture medium and diluent for immunoassay has high purity, no toxicity, no antigenicity and good thermal stability, and can be mixed with bovine serum albumin for use to enhance the sealing effect; the gel is a polymer solution or sol with a certain concentration, the viscosity is gradually increased under proper conditions, the fluidity is finally lost, the whole system becomes an elastic semi-solid with uniform appearance and a certain form is kept, the temperature is strictly controlled when the gel is blended with other liquids, the formula is regulated, the anti-aging agent is added to prevent the gel from generating, the gel is easily lost due to the lack of control, and other solutes in the solution are poorly dispersed. This material was therefore excluded as protein.
So that the bovine serum albumin is easier to control closed conditions, the preparation process is simpler and more convenient, and the bovine serum albumin has the functions of protecting the activity of the protein and stabilizing the conformation of the protein, thereby slowing down the trend of signal attenuation. In combination, the selection of bovine serum albumin works best.
Preferably, the surfactant is selected from one or more of Tween-20, tween-80, triton X100, CHAPS. Surfactants are compounds having a hydrophobic group and a hydrophilic group. The surfactant can cause colloid to be aggregated through a bridging mechanism, and further plays a certain role in promoting the sealing of latex microspheres. The present invention selects the surfactant CHAPS as one of the components of the capping reagent.
Preferably, the preservative is one or more selected from 1, 2-hexanediol, p-hydroxyacetophenone, sodium azide and ProClin300, wherein the 1, 2-hexanediol has a preservative effect when being added in about 5% because of good solubility, non-corrosiveness, antibacterial property and the like in the industries of medicines and the like, but the bacteriostatic effect of the component is more negative, so that whether the component is polluted during opening and use is not mastered, and the bacteriostatic effect of the product can not be compared with that of a real preservative for storage after the product is opened, so that the substance is excluded as the preservative. The p-hydroxyacetophenone has the advantages of no carcinogenicity and irritation, stronger antibacterial and antiseptic property, higher safety and the like, but the current obtaining way of the p-hydroxyacetophenone is not ideal: the production cost of the process is low but the production amount of byproducts is large; the other process has high product purity but high cost, and the substance is not suitable for being used as a preservative from the aspect of cost performance; sodium azide has excellent antiseptic and bactericidal properties, but sodium azide is irritating to the eyes and skin, such as inhalation, oral or percutaneous absorption, and can cause toxic death. And is particularly dangerous to use: explosion can occur when heated, exposed to open fire, or subjected to friction, shock, or impact. Since the present substance is too dangerous, it is also not suitable to choose it as a preservative; proClin300 preservative is a high-efficiency bacteriostatic agent, is commonly used in products such as in-vitro diagnosis of various reagents, quality control products, calibrator, buffer solution and the like, and has high-efficiency bacteriostatic effect on microorganisms. The broad-spectrum antibacterial activity, the excellent compatibility and stability and the low toxicity thereof under the use concentration are adopted, so that the product is harmless and healthy in the recommended use concentration, and has wide applicable PH range and good water solubility; the ProClin300 preservative can inhibit the growth of bacteria, fungi and yeasts for a longer time, so that the shelf life of the product is prolonged, and particularly, the ProClin300 preservative has no influence on the functions of most enzymes or antibody crosslinking reactions and the change of absorbance, so that the detection index is not interfered. The present materials are therefore suitable as preservatives in the present invention.
In conclusion, the preservative is the most suitable for ProClin300, and has the advantages of high cost performance, excellent anti-corrosion and antibacterial performance, safer use and the like. The final selected components of the blocking agent are therefore: purified water, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, bovine serum albumin,CHAPS、ProClin300。
The technical scheme of the invention has the following beneficial effects:
the invention provides a method for preparing an antibody coupled microsphere compound, which is applied to an antibody coupled microsphere compound, can reduce the nonspecific adsorption of an immune detection reagent, can improve the sensitivity, repeatability and stability of detection, and can improve the performance of the detection reagent, and an added stabilizer mainly serves as a blocking agent to play a role in stabilizing the conformation of the detection reagent, so that the trend of signal attenuation is slowed down; the PB buffer solution has good buffer effect and good salt balance capability, can play a role in regulating osmotic pressure, can maintain the activity of an antibody in the reagent in a short period, greatly improves the stability of the reagent, and can obtain good stability when the blocking agent is used for blocking an antibody coupling microsphere compound.
Drawings
FIG. 1 is a graph showing the stability of the test sample of example 1, comparative example 1 and comparative example 2 according to the present invention by adding different amounts of stabilizing agents.
Detailed Description
The invention will be further described with reference to the drawings and the specific examples.
Example 1:
the embodiment provides a blocking agent of an antibody coupled microsphere complex, which comprises the following blocking agent raw materials in percentage by weight: 97.8% PB (10 mM), 2% BSA, 0.05%0.05％CHAPS、0.1％ProClin300。
Comparative example 1:
the present comparative example 1 provides a blocking agent for an antibody-coupled microsphere complex comprising the following blocking agent raw materials in weight percent: 94.8% PB (10 mM), 5% BSA, 0.05%0.05％CHAPS、0.1％ProClin300。
Comparative example 2:
this comparative example 2 provides a blocking agent for an antibody-coupled microsphere complex comprising the following blocking agent raw materials in weight percent: 99.6% PB (10 mM), 0.2% BSA, 0.05%0.05％CHAPS、0.1％ProClin300。
Experimental example:
the experimental example prepares an immune turbidimetry antibody reagent:
(1) Antibody labeling: 300. Mu.L of 200nm latex microspheres were added to 50mM MES solution, activated by adding (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution, and a certain amount of antibody was added for coupling at room temperature for 2 hours, and after completion, unreacted reagent was removed by centrifugation.
(2) And (3) microsphere sealing: the antibody-conjugated latex microspheres were resuspended and dispersed in 15mL of blocking agent (example 1 and comparative examples 1-2), respectively, sonicated, and the blocking agent used was as shown in Table 1 below, and microsphere blocking was performed with the blocking agent formulated according to the following formulation, and the influence of the different components of the blocking agent on the sensitivity and precision of the detection reagent was examined:
TABLE 1
And (3) testing: taking prepared immune turbidimetric antibody reagent to test 0, 20, 100, 200, 400 and 600IU/mL of 6-concentration calibrator respectively, testing on a protein analysis instrument, performing five repeated tests on each concentration, calculating the difference and average value of the test reading reactivity, and detecting the following table 2, table 3 and figure 1:
TABLE 2
TABLE 3 Table 3
Referring to fig. 1, the result of example 1 shows an increasing curve with better linearity; antigen-antibody reactivity under otherwise identical preparation conditions: example 1> comparative example 2> comparative example 1, it is presumed that the BSA content of example 1 may be effective in reducing non-specific interference during immune reaction.
Through the verification of the experimental example, the blocking agent provided by the invention is applied to the antibody coupling microsphere compound, so that the nonspecific adsorption of an immunodetection reagent can be reduced, the sensitivity, repeatability and stability of detection can be improved, and the performance of the detection reagent can be improved.
As can be seen from the above examples 1, comparative examples 1-2 and experimental examples, the present invention provides an antibody-coupled microsphere complex, which can reduce the nonspecific adsorption of an immunodetection reagent, improve the sensitivity, repeatability and stability of detection, and improve the performance of the detection reagent, and the added stabilizer mainly acts as a blocking agent to stabilize the conformation thereof, thereby slowing down the tendency of signal attenuation; the PB buffer solution has good buffer effect and good salt balance capability, can play a role in regulating osmotic pressure, can maintain the activity of an antibody in the reagent in a short period, greatly improves the stability of the reagent, and can obtain good stability when the blocking agent is used for blocking an antibody coupling microsphere compound.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent structural changes made by the description of the present invention and the accompanying drawings or direct/indirect application in other related technical fields are included in the scope of the invention.
Claims (6)
1. The blocking agent of the antibody-coupled microsphere complex is characterized by comprising the following blocking liquid raw materials in percentage by weight: 70% -90% of buffer solution, 10% -30% of stabilizer, 0.01% -5% of surfactant and 0.1% -0.5% of preservative.
2. The blocking agent for antibody-coupled microsphere complexes according to claim 1, wherein the buffer is selected from one or more of PB buffer, tris-hydrochloric acid.
3. The blocking agent for antibody-coupled microsphere complexes according to claim 1, wherein the PB buffer material comprises purified water, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride.
4. The blocking agent for antibody-coupled microsphere complexes according to claim 1, wherein the stabilizing agent is selected from the group consisting of bovine serum albumin, casein, and combinations thereof,And one or more of gels.
5. The blocking agent for antibody-coupled microsphere complexes according to claim 1, wherein the surfactant is selected from one or more of tween-20, tween-80, triton X100, CHAPS.
6. The blocking agent for antibody-coupled microsphere complexes according to claim 1, wherein the preservative is selected from one or more of 1, 2-hexanediol, p-hydroxyacetophenone, sodium azide and ProClin 300.
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