CN116298267A - Sealing liquid for antigen coated reaction plate, application of sealing liquid and kit - Google Patents
Sealing liquid for antigen coated reaction plate, application of sealing liquid and kit Download PDFInfo
- Publication number
- CN116298267A CN116298267A CN202310540024.5A CN202310540024A CN116298267A CN 116298267 A CN116298267 A CN 116298267A CN 202310540024 A CN202310540024 A CN 202310540024A CN 116298267 A CN116298267 A CN 116298267A
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- antigen
- blocking solution
- reaction plate
- results
- coated reaction
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- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 235000013930 proline Nutrition 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 229940074410 trehalose Drugs 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- 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
-
- 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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention belongs to the field of biotechnology, and discloses a sealing liquid of an antigen coated reaction plate, application of the sealing liquid and a kit, wherein the sealing liquid comprises inorganic salt, a buffer, sodium caseinate, beta-cyclodextrin, BSA, trehalose, glycerol, an emulsifier and a preservative; in the blocking solution, the concentration of casein sodium, beta-cyclodextrin, BSA, trehalose and glycerol are respectively as follows: 1 to 5g/L, 0.05 to 0.2g/L, 5 to 20g/L and 0.1 to 1g/L. The sealing liquid of the antigen coated reaction plate can improve the stability of the antigen coated reaction plate and prolong the shelf life of the kit; improves the resistance to change of the reaction plate under the condition of transportation and storage, and is a sealing liquid for coating the reaction plate with the antigen with high efficiency and stability.
Description
Technical Field
The invention belongs to the technical field of antigen reaction plate coating, and particularly relates to a sealing liquid for an antigen coating reaction plate, application of the sealing liquid and a kit.
Background
An enzyme-linked immunosorbent assay (ELISA) is a common technique in immunological diagnosis, and the principle is to combine the high specificity of antigen-antibody reaction with the high-efficiency catalysis of enzyme to substrate, and to measure the Optical Density (OD) value of the colored substance produced by the enzymatic decomposition of the substrate by an enzyme-labeled instrument. The method has the advantages of strong specificity, high sensitivity, simple method, large analysis capacity, low detection cost and the like, and is widely applied without expensive instruments.
The antigen reaction plate is an important component of an enzyme-linked immunosorbent assay, is a place of the enzyme-linked immunosorbent assay, and is also a foundation stone for success or failure of the whole enzyme-linked immunosorbent assay. The preparation process includes physical adsorption of antigen or antibody onto the surface of solid carrier, sealing with sealing liquid, drying and packing.
The sealing liquid commonly used for the reaction plate at present mainly comprises skimmed milk powder, gelatin, albumin, saccharides and the like. The antigen reaction plate prepared by the sealing liquid can be placed for 6-8 months at the temperature of 2-8 ℃ and has obvious OD value degradation, obvious sensitivity degradation and incorrect detection result of clinical samples. Also, the kit often encounters repeated changes from low temperature to room temperature during transportation and use, which shortens the life of the antigen-reaction plate.
D1: CN115184620a provides a quantum dot fluorescent detection test strip of PLA2R antibody, a kit and application thereof, and the specification records: in the step 2), the quantum dot-labeled PLA2R antibody or the quantum dot-labeled chicken IgY is prepared by the following method: a: adding cadmium selenide quantum dots into MES buffer solution, and activating; then EDC and NHS are sequentially added into the reaction kettle, and then PLA2R antibody or chicken IgY antibody is added for light-shielding reaction; b: c, adding casein and glycine into the quantum dot marking liquid in the step a, and carrying out light-shielding stirring reaction at room temperature for sealing; c: c, centrifuging the marked mixed solution in the step b, and eluting by using PBS buffer solution as eluent;
in this scheme, casein and glycine are used for blocking.
D2: CN115327137A discloses a human immunoglobulin G4 subtype chemiluminescence immunoassay kit, which comprises an R1 reagent, an R2 reagent, an R3 reagent and a sample diluent, wherein the R1 reagent comprises a biotin-marked anti-human immunoglobulin G4 coated antibody and a first buffer solution, the first buffer solution comprises 50-500 mmol/L inorganic salt, 0.5-1 wt% of a blocking agent, 0.5-1 wt% of a surfactant, 0.1-0.5 wt% of a thickener, 1-5 wt% of saccharides, 0.1-0.5G/L of a blocking agent, 0.01-0.1 wt% of an antiseptic, 0.3wt% of an animal serum, the R2 reagent comprises a light-emitting marker-marked anti-human immunoglobulin G4 captured antibody and a second buffer solution, the second buffer solution comprises 50-500 mmol/L inorganic salt, 0.5-1 wt% of a blocking agent, 0.5-1 wt% of a surfactant, 0.1-0.5 wt% of a third buffer solution, 0.1-0.5 wt% of a surfactant, 0.1-0.5 wt% of a third buffer solution, 0.01-0.5 wt% of a protease, 0.01-0.1 wt% of a third buffer solution, 0.01-0.5 wt% of a protease, 0.1-0.5 wt% of a third buffer solution, and 0.01-0.1 wt% of a protease. The saccharides in the first buffer solution consist of sucrose and trehalose, and the mass ratio of the sucrose to the trehalose is (2-5): 1, a step of; the animal serum in the first buffer solution and the second buffer solution consists of fetal bovine serum, porcine serum and horse serum respectively, and the mass ratio of the fetal bovine serum to the porcine serum to the horse serum is (2-5): 1: (0.1 to 0.5);
the blocking agent is casein and/or bovine serum albumin.
D3: CN114703252a discloses a kit for detecting the content of hirudin, bivalirudin, dabigatran in blood plasma, comprising an R1 reagent, an R2 reagent, a dilution reagent, a hirudin calibrator, a bivalirudin calibrator and a dabigatran calibrator; the R1 reagent comprises thrombin, a first buffer solution and a first auxiliary material, the R2 reagent comprises a chromogenic substrate, a second buffer solution and a second auxiliary material, and the diluting reagent comprises a third buffer solution and a third auxiliary material; the hirudin calibrator comprises hirudin, the bivalirudin calibrator comprises bivalirudin, and the dabigatran calibrator comprises dabigatran;
the first auxiliary material comprises a first stabilizer, wherein the first stabilizer comprises one or more of sodium chloride, glycine, sucrose, galactose, polyethylene glycol 4000, span-40, gelatin, tween-20, trehalose, glucose, beta-cyclodextrin, mannitol and potassium chloride;
the hirudin calibrator comprises a first excipient and a first protective agent; the bivalirudin calibrator comprises a second excipient and a second protective agent; the dabigatran etexilate calibrator comprises a third excipient and a third protective agent; the first excipient, the second excipient and the third excipient are at least one of hydroxyethyl starch, glycine, mannitol, proline and casein;
d4: CN113341148A provides a rapid detection kit for simultaneously detecting sulfanilamide, florfenicol, tilmicosin and trimethoprim in pork, the kit comprises the following components: reaction liquid A, reaction liquid B, negative control liquid, concentrated cleaning agent, color developing agent A, color developing agent B, stopping agent, premixing pore plate and reaction pore plate; the reaction liquid A is a mixed working liquid of sulfonamide, florfenicol, tilmicosin and trimethoprim monoclonal antibodies; the mixed working solution is obtained by diluting the reaction solution A diluent; the formula of the reaction liquid A diluent per 1000ml is as follows: 100ml 0.5M EDTA,900ml 0.02M borate buffer, 0.05% cyclodextrin (0.5 g/L), 0.05% sodium caseinate (0.5 g/L), 0.10% Triton X-100,0.05% sodium azide, 0.10% trehalose (1 g/L), 0.05% polyethylene glycol, 0.05% polyacrylamide, 0.05% whey protein (0.5 g/L).
The above-mentioned reference document adopts specific antigen, absorption liquid or sealing liquid to make treatment, and its technical problem is how to raise detection accuracy.
D5: CN113267621B discloses a stabilizing agent for ELISA kit coated plate, a preparation method thereof, a kit coated plate and a kit. The stabilizing agent for the ELISA kit coated plate comprises water and the following components in percentage by mass: 5% -15% of glycerol, 0.5% -1.5% of trehalose, 0.005% -0.015% of sodium glutamate and 0.002% -0.008% of thioredoxin; the preparation method comprises the following steps: s1, uniformly mixing glycerol, trehalose, sodium glutamate and water, and regulating the pH value to 7 to obtain a mixed solution; s2, adding thioredoxin into the mixed solution in the step S1, and uniformly mixing to obtain the thioredoxin. The stabilizing agent for the ELISA kit coated plate can be used for preparing the ELISA kit coated plate, and the ELISA kit prepared by the stabilizing agent has the advantages of good stability and long storage period.
The description is as follows: because glycerol is adopted, the glycerol contains a large amount of hydroxyl groups, the glycerol is stable, a protective film can be formed on the surface of the antigen by adsorbing the glycerol on a coating plate, the protein structure of the antigen is protected, the deposition speed of the antigen-antibody can be accelerated in the subsequent detection step, and the binding probability of the enzyme-labeled antibody and the antigen is increased. In addition, trehalose can interact with the bound water of the protein molecules of the antigen, and has good protection effect. In addition, the sodium glutamate can improve the water holding capacity of antigen protein molecules, improve the water retention of the antigen protein molecules and further maintain the biological activity of antigens. The thioredoxin has good thermal stability, low molecular weight and good dispersion performance, can be dispersedly embedded between a glycerol protection film and antigen protein molecules, promotes reduction of other proteins through interaction of a semi-stereinic acid mercaptan-disulfide bond, thereby playing a good role in antioxidation, the thioredoxin is used as a hydrogen receptor to participate in redox reaction, the probability of damage of antigens caused by oxidative damage is greatly reduced, and the thioredoxin is compounded and cooperated with trehalose and glycerol to play a good role in protecting antibodies or antigens, so that the preservation period of the prepared ELISA kit is greatly prolonged, and the stability of test data of the ELISA kit is improved.
It can be seen that the use of a combination of polyols, trehalose, sodium glutamate, thioredoxin can improve the stability and shelf life of the kit.
The technical problem that the present case solves is: how to further increase the shelf life of the kit.
Disclosure of Invention
In view of the shortcomings of the prior art, a first object of the present invention is to provide a blocking solution with a stable antigen coated reaction plate. The invention provides a sealing liquid for an antigen coated reaction plate, which is suitable for sealing the antigen coated reaction plate in an enzyme-linked immunosorbent assay and can prolong the shelf life of the reaction plate.
A second object of the present invention is to provide a use of the sealing solution for antigen-coated reaction plates and a kit.
In order to achieve the first object, the present invention adopts the following technical scheme: an antigen coated reaction plate sealing liquid comprises inorganic salt, buffer agent, casein sodium, beta-cyclodextrin, BSA, trehalose, glycerol, emulsifying agent and preservative;
in the blocking solution, the concentration of casein sodium, beta-cyclodextrin, BSA, trehalose and glycerol are respectively as follows: 1 to 5g/L, 0.05 to 0.2g/L, 5 to 20g/L and 0.1 to 1g/L.
In the blocking solution of the antigen-coated reaction plate, the concentration of casein sodium, beta-cyclodextrin, BSA, trehalose and glycerol in the blocking solution are respectively as follows: 2-4 g/L, 0.05-0.15 g/L, 5-20 g/L and 0.3-0.8 g/L.
Further, the sealing liquid for the antigen coated reaction plate comprises the following components in parts by weight:
NaCl 0.1～0.5M；
KCl 1～5mM；
Na 2 HPO 4 ·12H 2 O 5～10mM；
KH 2 PO 4 0.5～2mM；
1-5 g/L of sodium caseinate;
beta-cyclodextrin 0.05-0.2 g/L;
BSA 5～20g/L；
trehalose 5-20 g/L;
0.1-1 g/L glycerol;
Tween-20 0.2～1mL/L；
Proclin-300 0.2～1mL/L。
further, the sealing liquid for the antigen coated reaction plate comprises the following components in parts by weight:
NaCl 0.1～0.15M；
KCl 2.5～3mM；
Na2HPO4·12H2O 7～9mM；
KH2PO4 1.3～1.6mM；
2-4 g/L of sodium caseinate;
beta-cyclodextrin 0.05-0.15 g/L;
BSA 5～20g/L；
trehalose 5-20 g/L;
0.3-0.8 g/L glycerol;
Tween-20 0.3～0.8mL/L；
Proclin-300 0.3～0.8mL/L。
further, the sealing liquid of the antigen coated reaction plate is prepared by the following steps:
weighing the components, and firstly adding NaCl, KCl, na 2 HPO 4 ·12H 2 O、KH 2 PO 4 Stirring and dissolving the components with distilled water, adding the rest components into the distilled water one by one, stirring, dissolving one by one, then fixing the volume with distilled water, and cooling and preserving at 2-8 ℃.
In order to achieve the second object, the invention adopts the following technical scheme: the application of the sealing liquid of the antigen coating reaction plate, wherein the application method of the sealing liquid in the antigen coating reaction plate comprises the following steps: adding a blocking solution into the reaction plate adsorbed with the antigen, and incubating for 4-6 hours at 25+/-3 ℃.
Further, the application of the sealing liquid of the antigen coating reaction plate comprises the following steps of: and (5) throwing away the sealing liquid and beating to dry, or freeze-drying the antigen coated reaction plate or blow-drying at room temperature, and placing the antigen coated reaction plate into an aluminum foil bag for sealing and preserving.
Meanwhile, the invention also discloses a kit, which comprises an antigen coating reaction plate treated by the sealing liquid.
In the kit, the antigen is one of foot-and-mouth disease type O, foot-and-mouth disease type A, african swine fever and epidemic diarrhea.
Compared with the prior art, the invention has the following beneficial effects:
the sealing liquid has the advantages of easily obtained raw materials, low preparation cost, simple use method and stable properties. When in use, the protective agent is not needed to be added, and the plate is not needed to be washed after the sealing is completed. The sealing liquid and the protective agent are combined into a whole, the process flow is simplified, and the production efficiency is improved. Practices show that the antigen reaction plate of the sealing liquid improves the anti-mass and anti-change capability of the reaction plate under the transportation and storage conditions, the storage period is more than 24 months, the OD value is not obviously degraded, and the sensitivity is not obviously degraded.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
2g/L sodium caseinate;
beta-cyclodextrin 0.05g/L;
BSA 5g/L；
trehalose 5g/L;
0.3g/L glycerol;
Tween-20 0.3mL/L；
Proclin-300 0.3mL/L。
example 2
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
2.5g/L sodium caseinate;
beta-cyclodextrin 0.1g/L;
BSA 10g/L；
trehalose 10g/L;
0.5g/L glycerol;
Tween-20 0.5mL/L；
Proclin-300 0.5mL/L。
example 3
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
4g/L of sodium caseinate;
beta-cyclodextrin 0.15g/L;
BSA 20g/L；
20g/L trehalose;
0.8g/L glycerol;
Tween-20 0.8mL/L；
Proclin-300 0.8mL/L。
the preparation method of the sealing liquid in examples 1 to 3 comprises the following steps:
weighing the components, and firstly adding NaCl, KCl, na 2 HPO 4 ·12H 2 O、KH 2 PO 4 Stirring and dissolving the components with distilled water, adding the rest components into the distilled water one by one, stirring, dissolving one by one, then fixing the volume to 1L with distilled water, and cooling and preserving at 2-8 ℃.
Comparative example 1
Commercial sealing protection liquid in market:
name: liquid Plate Sealer OEM;
cargo number: 160902-04;
brand: CANDOR Bioscience.
Comparative example 2
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
beta-cyclodextrin 0.05g/L;
BSA 7g/L；
trehalose 5g/L;
0.3g/L glycerol;
Tween-20 0.3mL/L；
Proclin-300 0.3mL/L。
comparative example 3
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
7g/L of sodium caseinate;
beta-cyclodextrin 0.05g/L;
trehalose 5g/L;
0.3g/L glycerol;
Tween-20 0.3mL/L；
Proclin-300 0.3mL/L。
comparative example 4
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
2g/L sodium caseinate;
beta-cyclodextrin 0.5g/L;
BSA 5g/L；
trehalose 5g/L;
0.3g/L glycerol;
Tween-20 0.3mL/L；
Proclin-300 0.3mL/L。
comparative example 5
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
0.5g/L sodium caseinate;
beta-cyclodextrin 0.01g/L;
BSA 5g/L；
trehalose 5g/L;
0.34g/L glycerol;
Tween-20 0.3mL/L；
Proclin-300 0.3mL/L。
comparative example 6
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
2g/L sodium caseinate;
beta-cyclodextrin 0.05g/L;
BSA 5g/L；
trehalose 5g/L;
glycerin 50g/L;
Tween-20 0.3mL/L；
Proclin-300 0.3mL/L。
comparative example 7
The sealing liquid for antigen coated reaction plate includes the following components:
NaCl 8g/L；
KCl 0.2g/L；
Na 2 HPO 4 ·12H 2 O 2.9g/L；
KH 2 PO 4 0.2g/L；
2g/L sodium caseinate;
beta-cyclodextrin 0.05g/L;
BSA 0.5g/L；
trehalose 5g/L;
0.3g/L glycerol;
Tween-20 0.3mL/L；
Proclin-300 0.3mL/L。
performance verification experiment:
comparison of examples of the first part with commercially available samples
The protective effect of the blocking solution was demonstrated by comparison tests of the blocking solutions of examples 1 to 3 with commercially available blocking solutions (comparative example 1). The specific method comprises the following steps:
the blocking solutions of examples 1 to 3 and the antigen-coated reaction plate treated with the commercially available blocking solution (comparative example 1) were stored at 2 to 8℃and 37℃respectively, and the test and detection of sensitivity and specificity of the kit for acceleration stability and real-time stability were performed.
The accelerated stability test is to take antigen coated reaction plates stored at the temperature of 2-8 ℃ and 37 ℃ and sample the antigen coated reaction plates on 1 day, 3 days, 7 days, 15 days, 21 days and 30 days respectively, perform sensitivity test and specificity test of an ELISA kit, and then compare test results and compare the protection effects of different sealing solutions; the real-time stability test is to sample the antigen coated reaction plate stored at 2-8 ℃ in 1 st month, 3 months, 6 months, 12 months, 18 months and 24 months respectively for detection of the sensitivity and specificity test of the kit.
1. Material
1.1 Examples 1 to 3 and comparative example 1 together produced antigen-coated reaction plates.
1.2 serum: african swine fever virus positive serum, african swine fever virus negative serum, porcine reproductive and respiratory syndrome virus positive serum, porcine pseudorabies virus positive serum, and E.coli BL21 (DE 3) positive serum.
2. Method of
2.1 sampling
The accelerated stability test is to take and put antigen coated reaction plates stored at the temperature of 2-8 ℃ and the temperature of 37 ℃ for sampling on the 1 st day, 3 days, 7 days, 15 days, 21 days and 30 days respectively;
the real-time stability test is to sample antigen coated reaction plates stored at 2-8 ℃ in 1 st month, 3 months, 6 months, 12 months, 18 months and 24 months respectively.
2.2 inspection
2.2.1 Kit sensitivity detection
The african swine fever virus positive serum was diluted with sample dilutions at dilution factors of 1:800, 1:1600, 1:3200, 1:6400, 1:12800 and 1:25600;
2.2.2 Kit specific detection
The sample dilutions were used to dilute african swine fever virus negative serum, porcine reproductive and respiratory syndrome virus positive serum, porcine pseudorabies virus positive serum, and escherichia coli BL21 (DE 3) positive serum, respectively, at a ratio of 1:100.
2.3 preparation of reagents
All reagents and samples were returned to 22-28 ℃ before use, and the reagents should be mixed by gentle rotation or shaking.
2.3.1 preparation of washing liquid
1 part of 20-time concentrated washing liquid is taken and added into 19 parts of double distilled water, and the mixture is uniformly mixed. The prepared washing liquid should be used up within 7 days.
2.3.2 preparation of substrate solutions
The substrate solution A (urea hydrogen peroxide solution) and the substrate solution B (TMB solution) are uniformly mixed in equal volumes, and are prepared according to the quantity detected by the test.
2.3.3 sample addition
Adding diluted samples; the serum to be tested was added to the coating plate at 100 μl/well, and 2 wells for each of the negative control serum and positive control serum were simultaneously set (positive and negative control serum was directly applied without dilution).
2.3.4 incubation
And (5) sealing the plates, and placing the plates in a temperature box at 22-28 ℃ for incubation for 30 minutes.
2.3.5 washing
The wells were discarded, 300 μl of wash solution was added to each well, washed 4 times, and finally patted dry.
2.3.6 addition of enzyme-labeled antibody
100 μl of enzyme-labeled antibody was added to each well at a working concentration.
2.3.7 incubation
And (5) sealing the plates, and placing the plates in a temperature box at 22-28 ℃ for incubation for 30 minutes.
2.3.8 Washing
The method is the same as 2.3.5.
2.3.9 color development
And adding 100 mu l of substrate solution into each hole, sealing the plates, and placing the plates in a 22-28 ℃ incubator to incubate for 10 minutes in a dark place.
2.3.10 terminate
100 μl of stop solution was added to each well, and after gentle shaking mixing, the OD450nm was read at a wavelength of 450nm (reading should be completed within 5 minutes after addition of stop solution).
2.3.11 Determination of
2.3.11.1 test conditions
Positive control OD 450nm Value > 0.6, negative control serum OD 450nm The value is less than 0.2, and the test is judged to be true.
2.3.11.2 calculating the S/P value
2.3.11.3 determination
When the S/P value of the sample to be detected is more than or equal to 0.3, judging the sample to be detected as positive; and the S/P value of the sample to be detected is less than 0.3, and the sample to be detected is judged as negative.
3 test results
3.1 accelerated stability test results
The acceleration stability test is carried out on 4 combined antigen coated reaction plates in total in the examples 1, 2 and 3 and the comparative example 1, and the results show that the OD value and the S/P value of the antigen coated reaction plates in the examples 1, 2 and 3 are not obviously degraded, and the degradation of the OD value and the S/P value is within 15 percent; the antigen coated reaction plate of comparative example 1 has obvious degradation of OD value and S/P value, and the degradation rate of OD value exceeds 20% after 3 rd day of placing at 37 ℃ and reaches more than 90% of degradation rate at 30 th day. According to the Arrhenius law, the antigen coated reaction plates in examples 1, 2 and 3 can be stably placed for at least more than 2 years under the environmental condition of 2-8 ℃. The results are shown in tables 1 to 15:
table 1 shows the results of the accelerated stability test OD values on day 1 of examples 1 to 3 and comparative example 1;
table 2 shows the results of the OD values of the acceleration stability tests on day 3 of examples 1 to 3 and comparative example 1;
table 3 shows the results of the OD values of the acceleration stability tests on day 7 of examples 1 to 3 and comparative example 1;
table 4 shows the results of the OD values of the 15 th day acceleration stability tests of examples 1 to 3 and comparative example 1;
table 5 shows the results of the OD values of the 21 st-day acceleration stability tests of examples 1 to 3 and comparative example 1;
table 6 shows the results of the OD values of the 30 th day acceleration stability tests of examples 1 to 3 and comparative example 1;
table 7 shows the results of the test of the S/P values of the acceleration stability on day 1 of examples 1 to 3 and comparative example 1;
table 8 shows the results of the test of the S/P values of the acceleration stability on day 3 of examples 1 to 3 and comparative example 1;
table 9 shows the results of the test of the S/P values of the acceleration stability on day 7 of examples 1 to 3 and comparative example 1;
table 10 shows the results of the test of the S/P values of the acceleration stability on day 15 of examples 1 to 3 and comparative example 1;
table 11 shows the results of the test for the S/P values of the acceleration stability on day 21 of examples 1 to 3 and comparative example 1;
table 12 shows the results of the test of the S/P values of the acceleration stability at 30 th day of examples 1 to 3 and comparative example 1;
table 13 shows the degradation of the OD values of the accelerated stability positive serum tests of examples 1 to 3 and comparative example 1;
table 14 shows the results of the accelerated stability positive serum test on days 1, 3 and 7 of examples 1 to 3 and comparative example 1;
table 15 shows the results of the accelerated stability positive serum test S/P values at 15, 21 and 30 days of examples 1 to 3 and comparative example 1;
TABLE 1 results of detection of OD values for acceleration stability of reaction plates of different schemes
TABLE 2 results of detection of OD values for acceleration stability of reaction plates of different schemes
TABLE 3 results of detection of OD values for acceleration stability of reaction plates of different schemes
TABLE 4 results of detection of OD values for acceleration stability of reaction plates of different schemes
TABLE 5 results of detection of OD values for acceleration stability of reaction plates of different schemes
TABLE 6 results of detection of OD values for acceleration stability of reaction plates of different schemes
TABLE 7 results of accelerated stability test for S/P values for different schemes of reaction plates
TABLE 8 results of accelerated stability detection of S/P values for different schemes of reaction plates
TABLE 9 results of accelerated stability test for S/P values for different schemes of reaction plates
TABLE 10 results of accelerated stability test S/P values for different schemes reaction plates
TABLE 11 results of accelerated stability detection of S/P values for reaction plates of different schemes
TABLE 12 results of accelerated stability test S/P values for different schemes reaction plates
TABLE 13 different protocol reaction plates accelerated stability positive serum detection OD degradation (%)
TABLE 14 results of accelerated stability positive serum detection S/P values for different protocol reaction plates
TABLE 15 results of accelerated stability positive serum detection S/P values for different protocol reaction plates
3.2 Results of real-time stability test:
the real-time stability test detection is carried out on 4 combined antigen coated reaction plates in total in the examples 1, 2 and 3 and the comparative example 1, and the results show that the OD value and the S/P value of the positive control are degraded within 8% after the antigen coated reaction plates in the examples 1, 2 and 3 are placed at the temperature of 2-8 ℃ for 24 months without obvious degradation; titers above the critical value of African swine fever virus positive serum (1:6400) degrade within 23%. The OD value and the S/P value of the antigen coated reaction plate of the comparative example 1 are obviously degraded, the degradation rate of the OD value exceeds 35% after the antigen coated reaction plate is placed at 2-8 ℃ for 6 months, and the sensitivity test is reduced by one titer. The results are shown in tables 16 to 23.
Table 16 shows the results of the real-time stability test of examples 1 to 3 and comparative example 1 at month 1;
table 17 shows the results of the real-time stability test of examples 1 to 3 and comparative example 1 at month 3;
table 18 shows the results of the real-time stability test of examples 1 to 3 and comparative example 1 at month 6;
table 19 shows the results of the real-time stability test of examples 1 to 3 and comparative example 1 at month 12;
table 20 shows the results of the real-time stability test of examples 1 to 3 and comparative example 1 at month 18;
table 21 shows the results of the real-time stability test of examples 1 to 3 and comparative example 1 at month 24;
table 22 shows degradation conditions of OD values of the positive serum in real time stability of examples 1 to 3 and comparative example 1;
table 23 shows the degradation conditions of the real-time stability positive serum S/P values of examples 1 to 3 and comparative example 1;
table 16 results of real-time stability test
Table 17 real-time stability test results
Table 18 real-time stability test results
Table 19 real-time stability test results
Table 20 real-time stability test results
Table 21 results of real-time stability test
Table 22 real-time stability of the reaction plates of different schemes degradation of positive serum OD values
Table 23 real-time stability of the reaction plates of different schemes Positive serum S/P degradation
Second part comparison of example 1 and comparative examples 2-5
The experimental conditions were the same as in the first section, and the experimental results were as follows:
3.1 accelerated stability test results
Reference tables 24-37;
table 24 shows the results of the OD values of the acceleration stability tests on day 1 of example 2 and comparative examples 2 to 5;
table 25 shows the results of the OD values of the 3 rd-day acceleration stability tests of example 2 and comparative examples 2 to 5;
table 26 shows the results of the OD values of the 7 th day acceleration stability tests of example 2 and comparative examples 2 to 5;
table 27 shows the results of the OD values of the 15 th day acceleration stability tests of example 2 and comparative examples 2 to 5;
table 28 shows the results of the OD values of the 21 st-day acceleration stability tests of example 2 and comparative examples 2 to 5;
table 29 shows the results of the OD values of the 30 th day acceleration stability tests of example 2 and comparative examples 2 to 5;
table 30 shows the results of the test for the S/P values of the acceleration stability on day 1 of example 2 and comparative examples 2 to 5;
table 31 shows the results of the test for the S/P values of the acceleration stability on day 3 of example 2 and comparative examples 2 to 5;
table 32 shows the results of the test for the S/P values of the acceleration stability on day 7 of example 2 and comparative examples 2 to 5;
table 33 shows the results of the test for the S/P values of the acceleration stability on day 15 of example 2 and comparative examples 2 to 5;
table 34 shows the results of the test for the S/P values of the acceleration stability on day 21 of example 2 and comparative examples 2 to 5;
table 35 shows the results of the OD values of the 30 th day acceleration stability tests of example 2 and comparative examples 2 to 5;
table 36 shows the degradation of the OD values of the acceleration stability of example 2 and comparative examples 2 to 5;
table 37 shows the results of S/P values of the accelerated stability test of African swine fever positive serum of example 2, comparative examples 2 to 5 on days 1, 3, 7, 15, 21 and 30;
table 24 results of accelerated stability test OD values
Table 25 results of accelerated stability test OD values
Table 26 results of accelerated stability test OD values
Table 27 results of accelerated stability test OD values
Table 28 results of accelerated stability test OD values
Table 29 results of accelerated stability test OD values
TABLE 30 acceleration stability test S/P value results
TABLE 31 acceleration stability test S/P value results
Table 32 acceleration stability test S/P value results
Table 33 acceleration stability test S/P value results
Table 34 acceleration stability test S/P value results
Table 35 acceleration stability test S/P value results
Table 36 accelerated stability degradation of OD values of each combination (%)
TABLE 37S/P value results of African swine fever positive serum acceleration stability test
As can be seen from the results of the second section, the stability of comparative examples 2-5 was significantly less than that of example 1 during the accelerated stability test.
Third section comparison of example 1 and comparative examples 6-7
The experimental conditions were the same as in the first section, and the experimental results were as follows:
3.1 accelerated stability test results
Reference tables 38-49;
table 38 shows the results of the OD values of the acceleration stability tests on day 1 of example 1 and comparative examples 6 and 7;
table 39 shows the results of the OD values of the acceleration stability tests on day 3 of example 1 and comparative examples 6 and 7;
table 40 shows the results of the OD values of the acceleration stability tests on day 7 of example 1 and comparative examples 6 and 7;
table 41 shows the results of the OD values of the acceleration stability tests on day 15 of example 1 and comparative examples 6 and 7;
table 42 shows the results of the OD values of the acceleration stability tests on day 21 of example 1 and comparative examples 6 and 7;
table 43 shows the results of the OD values of the acceleration stability tests on day 30 of example 1 and comparative examples 6 and 7;
table 44 shows the results of the acceleration stability test S/P values on days 1 and 3 of example 1 and comparative examples 6 and 7;
table 45 shows the results of the test for S/P values of the acceleration stability on days 7 and 21 of example 1 and comparative examples 6 and 7;
table 46 shows the results of the test for S/P values of the acceleration stability on days 21 and 30 of example 1 and comparative examples 6 and 7;
table 47 shows the degradation of the OD values of the respective combinations of the acceleration stability of example 1 and comparative examples 6 and 7;
table 48 shows the results of the S/P values of the African swine fever positive serum acceleration stability tests on days 1, 3 and 7 of example 1 and comparative examples 6 and 7;
table 49 shows the results of S/P values of the African swine fever positive serum acceleration stability tests on days 15, 21 and 30 of example 1 and comparative examples 6 and 7;
table 38 results of accelerated stability test OD values
Table 39 accelerated stability test OD results
Table 40 results of accelerated stability test OD values
Table 41 accelerated stability test OD results
Table 42 acceleration stability test OD results
Table 43 accelerated stability test OD results
Table 44 acceleration stability test S/P value results
Table 45 acceleration stability test S/P value results
Table 46 acceleration stability test S/P value results
TABLE 47 acceleration stability degradation of OD values for each combination (%)
Table 48 results of S/P value test for African swine fever positive serum acceleration stability
TABLE 49S/P value results of African swine fever positive serum acceleration stability test
As can be seen from the results of the third section, the stability of comparative examples 6-7 was significantly lower than that of example 1 during the accelerated stability test.
Conclusion:
the sealing liquid of the antigen-coated reaction plate can improve the stability of the antigen-coated reaction plate, prolong the shelf life of a kit and improve the anti-quality-changing capability of the reaction plate under the conditions of transportation and storage.
The applicant believes that this is achieved synergistically by following factors:
1. beta-cyclodextrin is a cyclic compound formed by combining 7 glucose residues through beta-1, 4-glycosidic bonds, and has strong coating capability; glycerol contains a large amount of hydroxyl groups and can also form a coating film; the applicant hypothesizes that: beta-cyclodextrin forms a framework of the coating film, and glycerol forms a main filling part of the coating film due to the hydrophilicity of the beta-cyclodextrin, so that the coating strength and the coating flexibility of the coating film can be obviously improved;
comparison of comparative examples 4 and 5 shows that the coating film is difficult to form a stable coating skeleton when the dosage of beta-cyclodextrin is too small, and the coating film is not easy to be filled with glycerol when the dosage of beta-cyclodextrin is too large; the end result is poor stability;
as can be seen from comparative example 6, in the case of excessive amounts of glycerin, the coating stability is rather disadvantageous, and the applicant speculates that the possible reason is that, in the formulation of the present invention, excessive glycerin would cause difficulty in dispersing other active ingredients and would be disadvantageous for emulsification of the emulsifier;
the applicant believes that forming a stable, thin, flexible coating film is a very important precondition for achieving long-term stability based on the formulation of the present invention;
2. as is clear from comparative examples 2, 3 and 7, sodium caseinate and BSA alone or in an amount of 1g/L or less cannot exert excellent effects;
casein, BSA are present in whey protein, where casein is about 80% and BSA is about 1.6%;
the mechanism by which sodium caseinate and BSA are used together to achieve improved stability is not known, but it is believed that reducibility is an important factor in protecting antigens, BSA is a very typical reducing agent for a kit, and has 17 disulfide bonds, and a sulfhydryl group, which has an active chemical reaction and an anti-redox effect; sodium caseinate itself has strong stability and can play the roles of a buffering agent and an emulsifying agent, and the applicant speculates that an important factor of the synergy of the sodium caseinate and the BSA is to improve the stability of the BSA in the system of the invention, so that the BSA can be stably combined with an antigen in a coating film to avoid the oxidation of the antigen;
trehalose has the function of interacting with the binding water of protein molecules of antigens as described in CN113267621B, and shows good protection effect.
Based on the above analysis, the applicant believes that a thinner and stable coating film, stably dispersed BSA, sodium caseinate exists between the film and the antigen, and that sodium caseinate can make BSA and antigen bind more uniformly and stably, and better exert the oxidation resistance of BSA.
Through the above effects, the purpose of long-term storage is achieved.
The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to, i.e. does not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (10)
1. The blocking solution of the antigen coated reaction plate is characterized by comprising inorganic salt, a buffering agent, sodium caseinate, beta-cyclodextrin, BSA, trehalose, glycerol, an emulsifying agent and a preservative;
in the blocking solution, the concentration of casein sodium, beta-cyclodextrin, BSA, trehalose and glycerol are respectively as follows: 1 to 5g/L, 0.05 to 0.2g/L, 5 to 20g/L and 0.1 to 1g/L.
2. The blocking solution for antigen-coated reaction plate according to claim 1, wherein in the blocking solution, the concentration of casein sodium, β -cyclodextrin, BSA, trehalose, glycerol is respectively: 2-4 g/L, 0.05-0.15 g/L, 5-20 g/L and 0.3-0.8 g/L.
3. The blocking solution for antigen-coated reaction plates according to claim 1, comprising the following components and contents thereof:
NaCl 0.1～0.5M；
KCl 1～5mM；
Na 2 HPO 4 ·12H 2 O 5～10mM；
KH 2 PO 4 0.5～2mM；
1-5 g/L of sodium caseinate;
beta-cyclodextrin 0.05-0.2 g/L;
BSA 5～20g/L；
trehalose 5-20 g/L;
0.1-1 g/L glycerol;
Tween-20 0.2～1mL/L；
Proclin-300 0.2～1mL/L。
4. a blocking solution for antigen coated reaction plates according to claim 3, comprising the following components and their contents:
NaCl 0.1～0.15M；
KCl 2.5～3mM；
Na 2 HPO 4 ·12H 2 O 7～9mM；
KH 2 PO 4 1.3～1.6mM；
2-4 g/L of sodium caseinate;
beta-cyclodextrin 0.05-0.15 g/L;
BSA 5～20g/L；
trehalose 5-20 g/L;
0.3-0.8 g/L glycerol;
Tween-20 0.3～0.8mL/L；
Proclin-300 0.3～0.8mL/L。
5. the blocking solution for antigen-coated reaction plates according to claim 1, wherein the blocking solution is prepared by the steps of:
weighing the components, and firstly adding NaCl, KCl, na 2 HPO 4 ·12H 2 O、KH 2 PO 4 Stirring and dissolving the components with distilled water, adding the rest components into the distilled water one by one, stirring, dissolving one by one, then fixing the volume with distilled water, and cooling and preserving at 2-8 ℃.
6. Use of a blocking solution for an antigen coated reaction plate according to any one of claims 1 to 5, wherein the blocking solution is used in an antigen coated reaction plate by: adding a blocking solution into the reaction plate adsorbed with the antigen, and incubating for 4-6 hours at 25+/-3 ℃.
7. The use of a blocking solution for antigen coated reaction plates according to claim 6, wherein the subsequent treatment of the antigen coated reaction plates with a blocking solution is: and (5) throwing away the sealing liquid and beating to dry, or drying the antigen coated reaction plate, and placing the antigen coated reaction plate into an aluminum foil bag for sealing and preserving.
8. The use of the blocking solution for antigen coated reaction plates according to claim 6, wherein the antigen is one of foot-and-mouth disease type O, foot-and-mouth disease type a, african swine fever, and epidemic diarrhea.
9. A kit comprising a reaction plate coated with an antigen treated with a blocking solution according to any one of claims 1 to 5.
10. The kit of claim 9, wherein the antigen is one of foot-and-mouth disease type O, foot-and-mouth disease type a, african swine fever, epidemic diarrhea.
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