CN111157721A - Coating liquid for improving stability of chlamydia pneumoniae antigen/mycoplasma antigen in immunochromatography reagent and preparation method thereof - Google Patents
Coating liquid for improving stability of chlamydia pneumoniae antigen/mycoplasma antigen in immunochromatography reagent and preparation method thereof Download PDFInfo
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- CN111157721A CN111157721A CN202010045060.0A CN202010045060A CN111157721A CN 111157721 A CN111157721 A CN 111157721A CN 202010045060 A CN202010045060 A CN 202010045060A CN 111157721 A CN111157721 A CN 111157721A
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
Abstract
The application relates to the field of biological reagents, in particular to a coating liquid for improving the stability of a chlamydia pneumoniae antigen/mycoplasma antigen in an immunochromatography reagent and a preparation method thereof. The coating solution comprises a buffer solution, wherein the buffer solution comprises: a first component for immobilizing chlamydia antigen or mycoplasma antigen on a nitrocellulose membrane, a stabilizer, an antioxidant, and a preservative. Wherein the first component facilitates the immobilization of the antigen on the nitrocellulose membrane; the stabilizer can improve the stability of mycoplasma antigen and chlamydia antigen; the antioxidant can prevent oxidation of mycoplasma antigen and chlamydia antigen; the preservative can play a role in preserving mycoplasma antigens and chlamydia antigens. The components in the coating liquid cooperate with each other to protect the stability of the antigen in the immunochromatography reagent.
Description
Technical Field
The application relates to the field of biological reagents, in particular to a coating liquid for improving the stability of a chlamydia pneumoniae antigen/mycoplasma antigen in an immunochromatography reagent and a preparation method thereof.
Background
Chlamydia pneumoniae and mycoplasma pneumoniae are common human respiratory pathogenic bacteria, and often cause acute respiratory infection, especially pharyngitis, nasosinusitis, bronchitis, pneumonia and other diseases.
The detection methods of the chlamydia pneumoniae and the mycoplasma pneumoniae comprise a pathogen isolation culture method, a direct antigen detection method, a nucleic acid amplification detection method, a serological detection method, an enzyme-linked immunosorbent assay (ELISA), an immunochromatography method, a Polymerase Chain Reaction (PCR) method and the like. Among these detection methods, serological detection methods are most commonly used because they are easy to obtain samples and convenient to detect. The immunochromatography rapid diagnosis technology is an important means of a serological detection method, and the technology utilizes an immunological principle, after a chlamydia pneumoniae/mycoplasma antibody in a sample is combined with a secondary antibody marked by a tracer, under the action of chromatography, the immune complex is specifically combined and aggregated with a chlamydia pneumoniae/mycoplasma antigen coated on a nitrocellulose membrane, and a reaction signal is displayed. Because the detection means needs to coat the Chlamydia pneumoniae/mycoplasma antigen on the nitrocellulose membrane and store the nitrocellulose membrane at room temperature, the stability of the antigen has great influence on the quality of the detection reagent besides depending on the purity of the Chlamydia pneumoniae/mycoplasma antigen, and if the stability of the antigen is not good, the accuracy of a detection result is directly influenced.
The existing scheme for improving the stability of the antigen is an antigen protective agent or diluent of broad spectrum or other antigen types, and is a detection method which is used for liquid phase reactions such as an enzyme-linked immunosorbent assay, a chemiluminescence assay, an immunoturbidimetry assay and the like and the reagent needs to be stored at 2-8 ℃.
The immunochromatography method needs to coat the chlamydia pneumoniae/mycoplasma antigen on a nitrocellulose membrane, dry and store at normal temperature, and has higher requirements on the stability of the antigen. The current technology does not provide a solution to the need for antigen stability for immunochromatographic platforms.
Disclosure of Invention
The purpose of the embodiments of the present application is to provide a coating solution for improving stability of a chlamydia pneumoniae antigen/mycoplasma antigen in an immunochromatography reagent and a preparation method thereof, which aim to improve stability of the chlamydia pneumoniae antigen/mycoplasma pneumoniae antigen.
In a first aspect, the present application provides a technical solution:
a coating solution for improving stability of chlamydia pneumoniae antigens/mycoplasma antigens in an immunochromatographic reagent, comprising: a buffer solution; the buffer solution comprises: a stabilizer, an antioxidant, a preservative and a first component for immobilizing a chlamydia antigen or a mycoplasma antigen on a nitrocellulose membrane; the first component includes an alcohol.
The first component of the alcohol in the coating solution provided by the embodiment of the application is beneficial to fixing the antigen on the nitrocellulose membrane; the stabilizer can improve the stability of mycoplasma antigen and chlamydia antigen; the antioxidant can prevent oxidation of mycoplasma antigen and chlamydia antigen; the preservative can play a role in preserving mycoplasma antigens and chlamydia antigens. The components in the coating liquid cooperate with each other to protect the stability of the antigen in the immunochromatography reagent.
In other embodiments of the present application:
the concentration of the buffer solution is 10-30 mM, and each 100ml of the buffer solution contains:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 0.5 to 2.5g of antioxidant and 0.02 to 0.04ml of preservative;
alternatively, the first and second electrodes may be,
the concentration of the above buffer was 20mM, and each 100ml of the buffer contained:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 1.5ml to 3ml of antioxidant and 0.02ml to 0.04ml of preservative.
Within the range of the mixture ratio, the stability of the coating liquid to the antigen is better.
In other embodiments herein, the first component is methanol or isopropanol.
By adding methanol or isopropanol into the coating liquid, the mycoplasma pneumoniae antigen and the chlamydia pneumoniae antigen can be effectively immobilized on the nitrocellulose membrane.
In other embodiments of the present application, the stabilizer is bovine serum albumin.
Bovine serum albumin is a globulin extracted from bovine serum, is in solid powder form and has no potential biological infection. By adding bovine serum albumin into the coating liquid, the stability of mycoplasma antigen and chlamydia antigen can be effectively improved, and no biological infection is generated.
In other embodiments herein, the antioxidant comprises at least one of TCEP, 2-mercaptoethanol, or DTT.
By adding at least one of TCEP, 2-mercaptoethanol or DTT into the coating liquid, the oxidation of mycoplasma antigen or chlamydia antigen can be effectively prevented, and the stability of the mycoplasma antigen or chlamydia antigen can be improved.
In other embodiments of the present application, the preservative is Proclin 300.
The Proclin300 preservative does not influence the combination of antigen and antibody, and can effectively play a role in preserving mycoplasma antigen and chlamydia antigen, thereby improving the stability of the mycoplasma antigen and the chlamydia antigen and enabling the mycoplasma antigen and the chlamydia antigen to be stably applied to immunochromatography reagents.
In other embodiments of the present application, the coating solution is used for improving stability of mycoplasma pneumoniae antigens in immunochromatography reagents, and each 100ml of the buffer solution further comprises:
1.5g～2.5g EDTA-2Na+。
EDTA-2Na+is a protease inhibitor, can effectively prevent antigen from hydrolysis, and the components in the coating liquid cooperate with each other to protect the stability of the antigen in the immunochromatography reagent.
In other embodiments of the present application:
the coating solution is used for improving the stability of the mycoplasma pneumoniae antigen in an immunochromatography reagent, and each 100ml of the buffer solution further comprises: 3g to 10g of trehalose or 5g to 10g of sucrose;
alternatively, the first and second electrodes may be,
the coating solution is used for improving the stability of the chlamydia pneumoniae antigen in an immunochromatography reagent, and each 100ml of the buffer solution further comprises: 3g to 5g of trehalose and 3g to 5g of sucrose.
Both sucrose and trehalose act to increase the thermal destruction stability of the antigen. The heat-labile stability of the antigen can be further improved by adding sucrose and/or trehalose to the coating solution. In a second aspect, the present application provides a technical solution:
a method for preparing the coating liquid for improving the stability of the Chlamydia pneumoniae antigen/Mycoplasma pneumoniae antigen in the immunochromatography reagent comprises the following steps:
uniformly mixing the buffer solution, the first component, the stabilizer, the antioxidant and the preservative;
wherein, 3ml to 5ml of the first component, 0.3g to 1.5g of the stabilizer, 0.5g to 2g of the antioxidant and 0.02ml to 0.04ml of the preservative are added into each 100ml of the buffer solution.
The coating solution prepared by the method can greatly increase the efficiency of the antigen solidified on the nitrocellulose membrane, prevent the antigen from being oxidized and hydrolyzed and improve the stability of the antigen in the immunochromatography reagent.
In other embodiments of the present application, the buffer is prepared by: dissolving 0.145-0.435 parts by weight of disodium hydrogen phosphate, 0.012-0.036 parts by weight of sodium dihydrogen phosphate and 0.85 parts by weight of sodium chloride in 100ml of deionized water.
In a third aspect, the present application provides a technical solution:
a reagent for detecting Mycoplasma pneumoniae/Chlamydia pneumoniae antibodies, which comprises the coating liquid for improving the stability of Chlamydia pneumoniae antigens/Mycoplasma antigens in an immunochromatographic reagent.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a graph showing the continuous trend of the weak positive quality control results of example 1 and comparative example 1 provided in the experimental example 1 of the present application, plotted according to the data in Table 1, with the abscissa representing the acceleration time and the ordinate representing the fluorescence signal value;
FIG. 2 is a graph showing a continuous trend of the results of the control of middle-sun properties of example 1 and comparative example 1 provided in the experimental example 1 of the present application with respect to time, plotted according to the data in Table 2, with the abscissa representing the acceleration time and the ordinate representing the fluorescence signal value;
FIG. 3 is a graph showing the continuous trend of the strong positive quality control results of example 1 and comparative example 1 provided in the experimental example 1 of the present application, plotted according to the data in Table 3, with the abscissa representing the acceleration time and the ordinate representing the fluorescence signal value;
FIG. 4 is a graph showing the continuous trend of the weak positive quality control results of example 4 and comparative example 2 over time according to the data in Table 4, wherein the abscissa is the acceleration time and the ordinate is the fluorescence signal value;
FIG. 5 is a graph showing the continuous trend of the results of the positive quality control products of example 4 and comparative example 2, which are provided in Experimental example 2 of the present application, with respect to the data shown in Table 5, with the abscissa representing the acceleration time and the ordinate representing the fluorescence signal value;
fig. 6 is a continuous trend of the results of the strong positive quality control products of example 4 and comparative example 2 provided in experimental example 2 of the present application, plotted according to the data in table 6, with the abscissa being the acceleration time and the ordinate being the fluorescence signal value.
Detailed Description
The embodiment of the application provides a coating solution for improving the stability of a chlamydia pneumoniae antigen/mycoplasma antigen in an immunochromatographic reagent, which comprises the following components in percentage by weight: a buffer comprising:
a first component for immobilizing chlamydia antigen or mycoplasma antigen on a nitrocellulose membrane, a stabilizer, an antioxidant, and a preservative. The first component is an alcohol.
The mycoplasma pneumoniae antigen and the chlamydia pneumoniae antigen have similar effects, are natural antigens coated on a nitrocellulose membrane in respective serological detection items, need to be added into a coating solution, and then are detected by adopting an immunochromatography method. The first component of the alcohol in the coating solution provided by the embodiment of the application is beneficial to fixing the antigen on the nitrocellulose membrane; the stabilizer can improve the stability of mycoplasma antigen and chlamydia antigen; the antioxidant can prevent oxidation of mycoplasma antigen and chlamydia antigen; the preservative can play a role in preserving mycoplasma antigens and chlamydia antigens. The components in the coating liquid cooperate with each other to protect the stability of the antigen in the immunochromatography reagent.
In some embodiments of the present application, the first component comprises methanol or isopropanol. By adding methanol or isopropanol into the coating liquid, the mycoplasma pneumoniae antigen and the chlamydia pneumoniae antigen can be effectively immobilized on the nitrocellulose membrane.
Further, the stabilizer is bovine serum albumin. Bovine serum albumin is a globulin extracted from bovine serum, is in solid powder form and has no potential biological infection. By adding bovine serum albumin into the coating liquid, the stability of mycoplasma antigen and chlamydia antigen can be effectively improved, and no biological infection is generated.
Further, the antioxidant comprises at least one of TCEP, 2-mercaptoethanol, or DTT.
By adding at least one of TCEP, 2-mercaptoethanol or DTT into the coating liquid, the oxidation of mycoplasma antigen or chlamydia antigen can be effectively prevented, and the stability of the mycoplasma antigen or chlamydia antigen can be improved.
TCEP, herein known by the name tris (2-carboxyethyl) phosphine. TCEP has good stability and solubility in aqueous solution, acidic solution and alkaline solution. The coating liquid is not oxidized in the air, so that the addition of TCEP in the coating liquid can effectively improve the antioxidant performance of the mycoplasma antigen or the chlamydia antigen and improve the stability of the mycoplasma antigen or the chlamydia antigen.
Further, the preservative is Proclin 300.
The Proclin300 preservative does not influence the combination of antigen and antibody, and can effectively play a role in preserving mycoplasma antigen and chlamydia antigen, thereby improving the stability of the mycoplasma antigen and the chlamydia antigen and enabling the mycoplasma antigen and the chlamydia antigen to be stably applied to immunochromatography reagents.
In some embodiments of the present application, the coating liquid includes: a buffer solution; the concentration of the buffer solution is 10-30 mM, and each 100ml of the buffer solution contains:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 0.5 to 2.5g of antioxidant and 0.02 to 0.04ml of preservative; or
The concentration of the buffer was 20mM, and per 100ml of buffer contained:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 1.5ml to 3ml of antioxidant and 0.02ml to 0.04ml of preservative.
Illustratively, when the antioxidant is selected to be TCEP or DTT, the TCEP or DTT is measured in mass units since it is generally solid. Therefore, the coating liquid has the following composition: concentration 20mM, per 100ml of buffer:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 0.5 to 2.5g of antioxidant and 0.02 to 0.04ml of preservative.
For example, in the above example: the coating liquid comprises the following components: concentration 20mM, per 100ml of buffer:
4ml of the first component; 1.0g of stabilizer; 2.0g of antioxidant and 0.03ml of preservative.
Further optionally, the coating liquid in the above example has a composition of: concentration 20mM, per 100ml of buffer:
3.5ml to 4.5ml of the first component; 0.4g to 1.2g of stabilizer; 0.6-2.4 g of antioxidant and 0.025-0.035 ml of preservative.
Illustratively, when the antioxidant is 2-mercaptoethanol, it is measured in units of volume since 2-mercaptoethanol is generally liquid. Therefore, the coating liquid has the following composition: concentration 20mM, per 100ml of buffer:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 1.5ml to 3ml of antioxidant and 0.02ml to 0.04ml of preservative.
For example, in the above example: the coating liquid comprises the following components: concentration 20mM, per 100ml of buffer:
3.5ml of the first component; 0.8g of stabilizer; 1.7ml of antioxidant and 0.025ml of preservative.
Further optionally, the coating liquid in the above example has a composition of: concentration 20mM, per 100ml of buffer:
3.2ml to 4.6ml of the first component; 0.32g to 1.48g of stabilizer; 1.52ml to 2.8ml of antioxidant and 0.024ml to 0.038ml of preservative.
In some embodiments of the present application, the coating solution described above can be used to improve the stability of mycoplasma pneumoniae antigens in immunochromatographic reagents. The coating liquid comprises:
concentration 20mM, per 100ml of buffer:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 0.5 to 2.5g of antioxidant and 0.02 to 0.04ml of preservative; 1.5 g-2.5 g EDTA-2Na+(ii) a 3g to 10g of trehalose or 5g to 10g of sucrose.
EDTA-2Na+The Chinese name of disodium edetate is a protease inhibitor which can effectively prevent antigen from hydrolysis, and all components in the coating liquid cooperate with each other to protect the stability of the antigen in the immunochromatography reagent.
Further optionally, the coating solution can be used for improving the stability of the mycoplasma pneumoniae antigen in the immunochromatographic reagent. The coating liquid comprises:
concentration 20mM, per 100ml of buffer:
3.6ml to 4.6ml of the first component; 0.3g to 1.0g of stabilizer; 1g to 2g of antioxidant and 0.02ml to 0.04ml of preservative; 1.5 g-2.5 g EDTA-2Na+(ii) a 3g to 10g of trehalose or 5g to 10g of sucrose. The antioxidant is TCEP.
Further optionally, the coating solution can be used for improving the stability of the mycoplasma pneumoniae antigen in the immunochromatographic reagent. The coating liquid comprises:
concentration 20mM, per 100ml of buffer:
3ml to 5ml of the first component; 0.3g to 1.0g of stabilizer; 1.5-2.5 g antioxidant and 0.02-0.04 ml preservative; 1.6 g-2.4 g EDTA-2Na+(ii) a 3g to 10g of trehalose or 5g to 10g of sucrose. The antioxidant is DTT. In some embodiments of the present application, the coating solution described above can be used to improve the stability of mycoplasma pneumoniae antigens in immunochromatographic reagents.
The coating liquid comprises:
concentration 20mM, per 100ml of buffer:
3ml to 5ml of the first component; 0.3g to 1.5g of stabilizer; 0.5 to 2.5g of antioxidant, 0.02 to 0.04ml of preservative and 1.5 to 2.5g of EDTA-2Na+。
Further optionally, the coating solution can be used for improving the stability of the mycoplasma pneumoniae antigen in the immunochromatographic reagent. The coating liquid comprises:
concentration 20mM, per 100ml of buffer:
3ml to 5ml of the first component; 0.3g to 1.0g of stabilizer; 1g to 2g of antioxidant, 0.02ml to 0.04ml of preservative and 1.5g to 2.5g of EDTA-2Na+。
In some embodiments of the present application, the coating solution described above can be used to improve the stability of the chlamydia pneumoniae antigen in the immunochromatographic reagent. The coating liquid comprises:
the concentration is 10-30 mM, and each 100ml of buffer solution contains:
3ml to 5ml of the first component; 0.5g to 1.5g of stabilizer; 0.5 to 1.2g of antioxidant, 0.02 to 0.04ml of preservative, 3 to 5g of trehalose and 3 to 5g of sucrose. The antioxidant is DTT.
In some embodiments of the present application, the coating solution described above can be used to improve the stability of the chlamydia pneumoniae antigen in the immunochromatographic reagent. The coating liquid comprises:
concentration 20mM, per 100ml of buffer:
3ml to 5ml of the first component; 0.5g to 1.5g of stabilizer; 1.5 to 3ml of antioxidant, 0.02 to 0.04ml of preservative, 3 to 5g of trehalose and 3 to 5g of sucrose. The antioxidant is 2-mercaptoethanol.
Some embodiments of the present application also provide a method for preparing the aforementioned coating solution for improving stability of chlamydia pneumoniae antigens/mycoplasma antigens in immunochromatographic reagents, comprising:
uniformly mixing the buffer solution, the first component, the stabilizer, the antioxidant and the preservative;
wherein, 3ml to 5ml of the first component, 0.3g to 1.5g of the stabilizer, 0.5g to 2g of the antioxidant and 0.02ml to 0.04ml of the preservative are added into each 100ml of the buffer solution.
Further, the concentration of the buffer solution is 10 to 30 mM. Optionally, the concentration of the buffer solution is 12 to 28 mM. Optionally, the concentration of the buffer solution is 14 to 26 mM. Further, the buffer solution is prepared by: dissolving 0.145-0.435 parts by weight of disodium hydrogen phosphate, 0.012-0.036 parts by weight of sodium dihydrogen phosphate and 0.85 parts by weight of sodium chloride in 100ml of deionized water.
Illustratively, the concentration of the above buffer is 20 mM. The above buffer solution is prepared by: the solution was prepared by dissolving 0.29 parts by weight of disodium hydrogen phosphate, 0.024 parts by weight of sodium dihydrogen phosphate, and 0.85 parts by weight of sodium chloride in 100ml of deionized water.
The coating solution prepared by the method can greatly increase the efficiency of the antigen solidified on the nitrocellulose membrane, prevent the antigen from being oxidized and hydrolyzed and improve the stability of the antigen in the immunochromatography reagent.
Some embodiments of the present application also provide a mycoplasma pneumoniae/chlamydia pneumoniae antibody detection reagent, including the coating solution for improving stability of chlamydia pneumoniae antigens/mycoplasma antigens in an immunochromatographic reagent provided by the previous embodiments.
Specifically, the mycoplasma pneumoniae/chlamydia pneumoniae antibody detection reagent can be applied to mycoplasma pneumoniae lgM antibody detection reagent (immunochromatography) and mycoplasma pneumoniae lgG antibody detection reagent (immunochromatography). Or the reagent for detecting antibodies against mycoplasma pneumoniae/chlamydia pneumoniae can be applied to reagent for detecting antibodies against chlamydia pneumoniae lgM (immunochromatography) and reagent for detecting antibodies against chlamydia pneumoniae lgG (immunochromatography).
The features and properties of the present invention are further described in detail below with reference to examples:
example 1
The coating solution for improving the stability of the mycoplasma pneumoniae antigen in the immunochromatography reagent provided by the embodiment is prepared by the following steps:
1) reagent manufacturers: sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and methanol were purchased from Guangzhou chemical reagent factories; trehalose was purchased from Nanjing Zhongnuo bioengineering, Inc.; bovine serum albumin, TCEP, EDTA-2Na+Proclin300 was purchased from sigma aldrich trade company ltd.
2) Preparing a mycoplasma pneumoniae antigen coating solution: weighing 0.29g of disodium hydrogen phosphate, 0.024g of sodium dihydrogen phosphate and 0.85g of sodium chloride, and dissolving in 100ml of deionized water to obtain a PBS buffer solution with the molar concentration of 20 mM; to 100ml of 20mM PBS buffer, 5g of trehalose, 3ml of methanol, 0.5g of bovine serum albumin, 2g of TCEP, and 2g of EDTA-2Na were added+And 0.02ml of Proclin300, and stirring until all the components are dissolved to obtain the mycoplasma pneumoniae antigen coating solution.
Example 2
The basic steps are the same as those of example 1, except that: 3ml of methanol was replaced by 3ml of isopropanol.
Example 3
The basic steps are the same as those of example 1, except that:
adding 3g of trehalose, 5ml of methanol, 0.3g of bovine serum albumin, 0.5g of TCEP, 1.5g of EDTA-2Na + and 0.04ml of Proclin300 into 100ml of 10mM PBS buffer solution, and stirring until all components are dissolved to obtain the mycoplasma pneumoniae antigen coating solution. Wherein the PBS buffer was prepared by dissolving 0.145 parts by weight of disodium hydrogenphosphate, 0.012 parts by weight of sodium dihydrogenphosphate and 0.85 parts by weight of sodium chloride in 100ml of deionized water.
Example 4
The basic steps are the same as those of example 1, except that:
adding 3g of sucrose, 4ml of methanol, 1.5g of bovine serum albumin, 2.5g of TCEP, 2.5g of EDTA-2Na + and 0.03ml of Proclin300 into 100ml of 30mM PBS buffer solution, and stirring until all components are dissolved to obtain the mycoplasma pneumoniae antigen coating solution. Wherein the PBS buffer was prepared by dissolving 0.435 parts by weight of disodium hydrogenphosphate, 0.036 parts by weight of sodium dihydrogenphosphate, and 0.85 parts by weight of sodium chloride in 100ml of deionized water.
Comparative example 1
Providing a common coating liquid, which is prepared by the following steps:
weighing 0.29g of disodium hydrogen phosphate, 0.024g of sodium dihydrogen phosphate and 0.85g of sodium chloride, and dissolving in 100ml of deionized water to obtain a PBS buffer solution with the molar concentration of 20 mM; 5g of trehalose and 0.02ml of Proclin300 are added into 100ml of 20mM PBS buffer solution, and the mixture is stirred until all components are dissolved, so that the common antigen coating solution is obtained.
Experimental example 1
Mycoplasma pneumoniae was coated with the coating solutions provided in example 1, example 2 and comparative example 1, and then the stability of Mycoplasma was examined. The method comprises the following specific steps:
1) coating of mycoplasma pneumoniae: the Mycoplasma pneumoniae antigen (purchased from Meridian Life Science, cat. No. R01701) was diluted with the coating solutions provided in example 1, example 2 and comparative example 1, respectively, to a final concentration of 1.0mg/ml after dilution. The membranes were coated on nitrocellulose membranes (purchased from Sartorius, model CN95) with a gold spraying membrane-scribing instrument (BioDot, model XYZ3210) with a membrane-scribing parameter of 1.0 ul/cm. After coating, the coated film was dried in an air-blast drying oven (model DHG-9053A, Shanghai sperm macroexperimental facilities, Ltd., Equipment manufacturer) at 37 ℃ for 24 hours.
2) Preparing an immunochromatography reagent card: the three mycoplasma pneumoniae antigen coating films are respectively assembled with a marker combination pad (anti-human secondary antibody marked by fluorescent microspheres), absorbent paper and a PVC plate to form two groups of reagent cards.
3) Accelerated destruction of reagent cards: dividing the three groups of reagent cards into 5 parts by using aluminum foil bags respectively, and sealing the bags. Four of the above-mentioned materials were put in a constant temperature incubator (model GNP-9050, Shanghai sperm macroexperimental facilities Co., Ltd., Equipment Co., Ltd.) to accelerate destruction at 37 ℃ for 0 week, 1 week, 2 weeks, 3 weeks, and 4 weeks.
4) And (3) testing a reagent card: and (3) detecting each group of the reagent cards accelerated for 0 week, 1 week, 2 weeks, 3 weeks and 4 weeks by using a quality control product (self-made by enterprises), and obtaining the fluorescence detection values of the three groups of the reagent cards at normal temperature and different acceleration times.
The results are shown in Table 1-3 and attached figures 1-3:
TABLE 1 Weak Positive quality control results
Positive quality control results in table 2:
TABLE 3 Strong Positive quality control results
The above results show that after accelerated destruction in an environment at 37 ℃, fluorescence signal values of samples of examples 1 and 2 after accelerated destruction for four weeks are reduced within 10% when different concentrations of samples are detected, and fluorescence signal values of comparative example 1 after accelerated destruction for four weeks are reduced by about 20%, which indicates that the coating solution provided by the embodiment of the present application can improve the stability of the used mycoplasma pneumoniae antigen in the reagent for measuring mycoplasma pneumoniae antibodies by immunochromatography. When samples with different concentrations are detected, the consistency of detection results is better maintained.
The results of examples 3 and 4 are similar to those of examples 1 and 2, and the stability of the mycoplasma pneumoniae antigen can be effectively improved.
Example 5
The coating solution for improving the stability of the chlamydia pneumoniae antigen in the immunochromatographic reagent provided by the embodiment is prepared by the following steps:
1) reagent manufacturers: sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, methanol and sucrose were purchased from Guangzhou chemical reagent factory; trehalose was purchased from Nanjing Zhongnuo bioengineering, Inc.; bovine serum albumin, 2-mercaptoethanol, Proclin300 were purchased from sigma aldrich trade ltd.
2) Preparing a chlamydia pneumoniae antigen coating solution: weighing 0.29g of disodium hydrogen phosphate, 0.024g of sodium dihydrogen phosphate and 0.85g of sodium chloride, and dissolving in 100ml of deionized water to obtain a PBS buffer solution with the molar concentration of 20 mM; adding 5g of trehalose, 5g of sucrose, 3ml of methanol, 0.5g of bovine serum albumin, 3ml of 2-mercaptoethanol and 0.02ml of Proclin300 into 100ml of 20mM PBS buffer solution, and stirring until all components are dissolved to obtain the chlamydia pneumoniae antigen coating solution.
Example 6
The basic steps are the same as those of example 5, except that: the first component added was 3ml of isopropanol.
Example 7
The basic steps are the same as those of example 5, except that:
adding 3g of trehalose, 3g of sucrose, 5ml of methanol, 1.5g of bovine serum albumin, 1.5ml of 2-mercaptoethanol and 0.04ml of Proclin300 into 100ml of 25mM PBS buffer solution, and stirring until all components are dissolved to obtain the chlamydia pneumoniae antigen coating solution. Wherein the PBS buffer was prepared by dissolving 0.3625 parts by weight of disodium hydrogenphosphate, 0.03 parts by weight of sodium dihydrogenphosphate and 0.85 parts by weight of sodium chloride in 100ml of deionized water.
Example 8
The basic steps are the same as those of example 5, except that:
adding 4g of trehalose, 4g of sucrose, 4ml of methanol, 1.0g of bovine serum albumin, 2.0ml of 2-mercaptoethanol and 0.03ml of Proclin300 into 100ml of 25mM PBS buffer solution, and stirring until all components are dissolved to obtain the chlamydia pneumoniae antigen coating solution. Wherein the PBS buffer was prepared by dissolving 0.3625 parts by weight of disodium hydrogenphosphate, 0.03 parts by weight of sodium dihydrogenphosphate and 0.85 parts by weight of sodium chloride in 100ml of deionized water.
Comparative example 2
Providing a common coating liquid, which is prepared by the following steps:
weighing 0.29g of disodium hydrogen phosphate, 0.024g of sodium dihydrogen phosphate and 0.85g of sodium chloride, and dissolving in 100ml of deionized water to obtain a PBS buffer solution with the molar concentration of 20 mM; adding 5g of trehalose and 0.02ml of Proclin300 into 100ml of 20mM PBS buffer solution, and stirring until all components are dissolved to obtain the common antigen coating solution.
Experimental example 2
The chlamydia pneumoniae was coated with the coating solutions provided in example 5, example 6 and comparative example 2, and then the stability of the chlamydia was examined. The method comprises the following specific steps:
1) coating with chlamydia pneumoniae: the Chlamydia pneumoniae antigen (purchased from Meridian Life Science, cat # R02620) was diluted with a Chlamydia pneumoniae antigen coating solution and a common coating solution, respectively, to a final concentration of 1.0 mg/ml. The membranes were coated on nitrocellulose membranes (purchased from Sartorius, model CN95) with a gold spraying membrane-scribing instrument (BioDot, model XYZ3210) with a membrane-scribing parameter of 1.0 ul/cm. After coating, the coated film was dried in an air-blast drying oven (model DHG-9053A, Shanghai sperm macroexperimental facilities, Ltd., Equipment manufacturer) at 37 ℃ for 24 hours.
2) Preparing an immunochromatography reagent card: the three kinds of chlamydia pneumoniae antigen coating films are respectively assembled with a marker combination pad (anti-human secondary antibody marked by fluorescent microspheres), absorbent paper and a PVC plate to form three groups of reagent cards.
3) Accelerated destruction of reagent cards: dividing the three groups of reagent cards into 5 parts by using aluminum foil bags respectively, and sealing the bags. Four of the above-mentioned materials were put in a constant temperature incubator (model GNP-9050, Shanghai sperm macroexperimental facilities Co., Ltd., Equipment Co., Ltd.) to accelerate destruction at 37 ℃ for 0 week, 1 week, 2 weeks, 3 weeks, and 4 weeks.
4) And (3) testing a reagent card: and (3) detecting each group of the reagent cards accelerated for 0 week, 1 week, 2 weeks, 3 weeks and 4 weeks by using a quality control product (self-made by enterprises), and obtaining the fluorescence detection values of the three groups of the reagent cards at normal temperature and different acceleration times.
The results are shown in tables 4-6 and figures 4-6:
TABLE 4 Weak-positive quality control results
Results of positive quality control in Table 5
TABLE 6 Strong Positive results of quality control
The above results show that after accelerated destruction in an environment of 37 ℃, the fluorescence signal values of the samples of examples 5 and 6 after accelerated destruction for four weeks are reduced within 10% when different concentrations of the samples are detected, and the fluorescence signal values of the samples of comparative example 2 after accelerated destruction for four weeks are reduced by more than 30%, which indicates that the stability of the chlamydia pneumoniae antigen used in the reagent for measuring the chlamydia pneumoniae antibody by the immunochromatography method can be improved. When samples with different concentrations are detected, the consistency of detection results is better maintained.
The detection results of the embodiments 7 and 8 are similar to those of the embodiments 5 and 6, and the stability of the mycoplasma pneumoniae antigen can be effectively improved.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A coating solution for improving stability of chlamydia pneumoniae antigens/mycoplasma antigens in an immunochromatographic reagent, comprising: a buffer solution; the buffer solution comprises: a stabilizer, an antioxidant, a preservative and a first component for immobilizing a chlamydia antigen or a mycoplasma antigen on a nitrocellulose membrane; the first component includes an alcohol.
2. The coating solution for improving the stability of Chlamydia pneumoniae antigen/Mycoplasma pneumoniae antigen in an immunochromatographic reagent according to claim 1,
the concentration of the buffer solution is 10-30 mM, and each 100ml of the buffer solution contains:
3ml to 5ml of the first component; 0.3g to 1.5g of the stabilizer; 0.5 to 2.5g of the antioxidant and 0.02 to 0.04ml of the preservative;
alternatively, the first and second electrodes may be,
the concentration of the buffer was 20mM, and per 100ml of the buffer:
3ml to 5ml of the first component; 0.3g to 1.5g of the stabilizer; 1.5-3 ml of the antioxidant and 0.02-0.04 ml of the preservative.
3. The coating solution for improving the stability of Chlamydia pneumoniae antigen/Mycoplasma pneumoniae antigen in an immunochromatographic reagent according to claim 1,
the first component is methanol or isopropanol.
4. The coating solution for improving the stability of Chlamydia pneumoniae antigen/Mycoplasma pneumoniae antigen in an immunochromatographic reagent according to claim 1,
the stabilizer is bovine serum albumin.
5. The coating solution for improving the stability of Chlamydia pneumoniae antigen/Mycoplasma pneumoniae antigen in an immunochromatographic reagent according to claim 1,
the antioxidant comprises at least one of TCEP, 2-mercaptoethanol, or DTT.
6. The coating solution for improving the stability of Chlamydia pneumoniae antigen/Mycoplasma pneumoniae antigen in an immunochromatographic reagent according to claim 1,
the preservative is Proclin 300.
7. The coating solution for improving stability of Chlamydia pneumoniae antigen/Mycoplasma pneumoniae antigen in an immunochromatographic reagent according to any one of claims 2-6, wherein the coating solution is used for improving stability of Mycoplasma pneumoniae antigen in the immunochromatographic reagent, and further comprises per 100ml of the buffer solution:
1.5g～2.5g EDTA-2Na+。
8. the coating solution for improving the stability of Chlamydia pneumoniae antigens/Mycoplasma antigens in immunochromatography reagents according to any one of claims 2 to 6,
the coating solution is used for improving the stability of the mycoplasma pneumoniae antigen in an immunochromatography reagent, and each 100ml of the buffer solution further comprises: 3g to 10g of trehalose or 5g to 10g of sucrose;
alternatively, the first and second electrodes may be,
the coating solution is used for improving the stability of the Chlamydia pneumoniae antigen in an immunochromatography reagent, and further comprises per 100ml of the buffer solution: 3g to 5g of trehalose and 3g to 5g of sucrose.
9. A method for preparing a coating solution for improving the stability of a Chlamydia pneumoniae antigen/Mycoplasma antigen in an immunochromatographic reagent, comprising:
uniformly mixing the buffer solution, the first component, the stabilizer, the antioxidant and the preservative;
wherein, 3ml to 5ml of the first component, 0.3g to 1.5g of the stabilizer, 0.5g to 2g of the antioxidant and 0.02ml to 0.04ml of the preservative are added into each 100ml of the buffer solution.
10. A reagent for detecting mycoplasma pneumoniae/chlamydia pneumoniae antibodies, which comprises the coating solution according to any one of claims 1 to 8 for improving the stability of chlamydia pneumoniae antigens/mycoplasma antigens in an immunochromatographic reagent.
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