CN114324854B - Composite biological blocking agent and preparation method and reagent strip - Google Patents
Composite biological blocking agent and preparation method and reagent strip Download PDFInfo
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- CN114324854B CN114324854B CN202111508326.1A CN202111508326A CN114324854B CN 114324854 B CN114324854 B CN 114324854B CN 202111508326 A CN202111508326 A CN 202111508326A CN 114324854 B CN114324854 B CN 114324854B
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Abstract
The invention discloses a composite biological blocker for immunochromatography detection, a preparation method thereof and a reagent strip, wherein the blocker comprises a blocker buffer solution, wherein the blocker buffer solution is diluted with a mouse monoclonal antibody IgG-1, a mouse monoclonal antibody IgG-2, a goat anti-human IgM/RF polyclonal antibody and a rabbit polyclonal antibody IgG, and BSA is also added into the blocker buffer solution, wherein the blocker buffer solution is a Tris (hydroxymethyl) aminomethane-Tris-HCl buffer solution or a phosphate PBS buffer solution. The blocking agent can effectively reduce or eliminate the adverse effect of endogenous interfering substances on fluorescence immunochromatography detection, prevent false positive and false negative detection results, and can effectively prevent and control the influence of endogenous interfering substances in a sample on the immunochromatography detection after mixing a plurality of different mouse monoclonal antibodies, sheep polyclonal antibodies and rabbit polyclonal antibodies, thereby improving the accuracy of the detection results of the immunochromatography quantitative detection reagent.
Description
Technical Field
The invention relates to the technical field of immunochromatography detection, in particular to a composite biological blocker for immunochromatography detection, a preparation method thereof and a reagent strip.
Background
With the rapid development of the instant test (Point of care test, POCT) technology, hospitals and health offices can also provide accurate, sensitive, convenient and fast diagnosis and detection services for people.
Immunochromatography (immunochromatography assay) is a novel immunoassay mode which appears in the early 80 s of the 20 th century, and is a simple and rapid immunological detection technology based on immunofiltration (immunofiltrationassay). The principle is that a specific antibody is fixed on a certain zone of a nitrocellulose membrane, when one end of the dried nitrocellulose is immersed in a sample, the sample moves forward along the membrane due to capillary action, and when the sample moves to the zone fixed with the antibody, the corresponding antigen in the sample is specifically combined with the antibody, and the labeled colloidal gold or fluorescent substance can enable the zone to display certain light under specific excitation light, so that specific immunodiagnosis is realized. The technology has good sensitivity and specificity, is simple to operate, does not need expensive detection equipment, and enables the immunochromatography technology to be rapidly developed in the aspect of instant detection.
However, studies have shown that more than 3% of healthy individuals contain endogenous interfering factors, mainly including endogenous interfering substances such as antibodies to xenotropins (HA), antibodies to human animals (HAAA), and Rheumatoid Factors (RF), and that the proportion of those raised pets and treated with antibodies is higher. The effective means for reducing and eliminating endogenous interference is an important research direction for guaranteeing stable and reliable immune test results.
Commercially available blocking agents in the market at present mainly comprise HBR, TRUBlock, MAK and the like, and the blocking agent is added into a sample pad or a binding pad treatment liquid to treat glass fibers, and can be combined with interfering substances, so that the interference is reduced or even eliminated. However, because the types and the concentrations of the interfering substances in the sample are different, the influences of the interfering substances on different detection items are different, and the commercial blocker cannot completely prevent the interference of endogenous factors on the detection result.
Therefore, it is necessary to develop a blocker that prevents the interference of endogenous factors with the detection result.
Disclosure of Invention
The invention aims to solve the technical problem of providing a composite biological blocker for immunochromatography detection, which comprises a plurality of animal immunoglobulins interfering with a xenotropic antibody, an anti-animal antibody, an anti-rheumatoid factor and the like, has a good blocking effect on complex endogenous interfering factors, and can effectively reduce false positive or false negative results of immunochromatography detection caused by endogenous interfering substances.
In order to solve the technical problems, the invention adopts the technical scheme that the composite biological blocker for immunochromatography detection comprises a blocker buffer solution, wherein the blocker buffer solution is diluted with a mouse monoclonal antibody IgG-1, a mouse monoclonal antibody IgG-2, a sheep anti-human IgM/RF polyclonal antibody and a rabbit polyclonal antibody IgG;
Wherein the blocker buffer solution is Tris-HCl buffer solution or phosphate PBS buffer solution.
The composite biological blocker for immunochromatography detection in the technical scheme can effectively reduce or eliminate adverse effects of endogenous interference substances on fluorescence immunochromatography detection, prevent false positive and false negative detection results, and can effectively prevent and control the influences of endogenous interference substances in samples on the immunochromatography detection after mixing a plurality of different mouse monoclonal antibodies, sheep polyclonal antibodies and rabbit polyclonal antibodies, thereby improving the accuracy of detection results of immunochromatography quantitative detection reagents.
Preferably, the blocker buffer is 10mM Tris-HCl buffer or 10mM phosphate PBS buffer.
Preferably, the pH of the blocker buffer is 7.4.+ -. 0.2.
Preferably, the murine monoclonal antibody IgG-1, murine monoclonal antibody IgG-2, goat anti-human IgM/RF polyclonal antibody and rabbit polyclonal antibody IgG are diluted to 10mg/mL, 2mg/mL, respectively, with the blocker buffer.
Preferably, the blocker buffer is added to 10mg/mL BSA.
Preferably, proclin 300 is also added to the blocker buffer.
The invention provides a preparation method of a composite biological blocker for immunochromatography detection, which comprises the following steps:
s1, preparing a blocker buffer solution, wherein the blocker buffer solution adopts 10mM Tris-HCl buffer solution or 10mM PBS buffer solution, and the pH value of the blocker buffer solution is 7.4+/-0.2;
S2, respectively diluting the murine monoclonal antibody IgG-1, the murine monoclonal antibody IgG-2, the goat anti-human IgM/RF polyclonal antibody and the rabbit polyclonal antibody IgG to 10mg/mL, 2mg/mL and 2mg/mL by using the blocker buffer solution in the step S1;
s3, adding BSA to 10mg/mL of the blocker buffer solution in the step S2;
s4, adding Proclin 300 into the blocker buffer solution in the step S3 to obtain the composite biological blocker.
The invention provides a fluorescent immunodetection reagent strip, which comprises a sample pad, a binding pad, a coated nitrocellulose membrane and absorbent paper, wherein the sample pad is treated by a sample pad treatment liquid, and the sample pad treatment liquid comprises a buffer solution with pH of 8-10, a surfactant, trehalose, erythrocyte monoclonal antibody and the composite biological blocker according to the volume ratio of 0.5 percent.
Preferably, the preparation method of the bonding pad comprises (1) activating fluorescent microspheres and EDC in phosphate buffer, (2) adding detection antibody I and chicken IgY into the fluorescent microsphere solution for reaction, (3) performing a blocking reaction on the coupled microsphere-antibody complex, and centrifuging to obtain an immunofluorescent microsphere marker, (4) coating the immunofluorescent microsphere marker solution with a prepared concentration on glass fibers, and drying to obtain the bonding pad.
Preferably, the preparation method of the nitrocellulose membrane comprises the steps of preparing a T line solution by a detection antibody II, preparing a C line solution by a sheep anti-chicken IgY, coating the T detection line and the C quality control line area on the nitrocellulose membrane, and drying.
Preferably, the preparation method of the reagent strip comprises the steps of sequentially adhering a sample pad, a bonding pad, a coated nitrocellulose membrane and absorbent paper to a bottom plate from a sample adding end, and then cutting the sample pad, the bonding pad, the coated nitrocellulose membrane and absorbent paper to required widths to form the test strip.
After forming the test strip, the test strip is put into a plastic card shell to form the reagent card.
Drawings
The following is a further detailed description of embodiments of the invention with reference to the accompanying drawings:
FIG. 1 is a graph showing the correlation between the detection result and the target value of the experimental group (blocker addition) in the example;
FIG. 2 is a graph showing the correlation between the detection result and the target value of the control group (no blocking agent added) in the example.
Detailed Description
The invention relates to a composite biological blocker for immunochromatography detection, which comprises a blocker buffer solution, wherein a murine monoclonal antibody IgG-1, a murine monoclonal antibody IgG-2, a goat anti-human IgM/RF polyclonal antibody and a rabbit polyclonal antibody IgG are diluted in the blocker buffer solution, BSA is also added in the blocker buffer solution, the BSA is added to 10mg/mL in the blocker buffer solution, proclin 300 (the manufacturer is Sigma in the U.S., the product number is 48912-U), and the blocker buffer solution is a high-efficiency biological preservative, and the adding amount is one thousandth of the volume ratio and can be omitted;
Wherein the blocking agent buffer solution is Tris (hydroxymethyl) aminomethane-HCl buffer solution or phosphate PBS buffer solution, and the manufacturers of the murine monoclonal antibody IgG-1, the murine monoclonal antibody IgG-2, the goat anti-human IgM/RF polyclonal antibody and the rabbit polyclonal antibody are Nanjing Xuan biotechnology limited company, and the corresponding goods numbers are the murine monoclonal antibody IgG-1-5016M, the murine monoclonal antibody IgG-2-5017M, the goat anti-human IgM/RF polyclonal antibody-5030P and the rabbit polyclonal antibody IgG-5012N.
The blocking agent buffer solution is 10mM Tris-HCl buffer solution or 10mM phosphate PBS buffer solution, and the pH value is 7.4+/-0.2.
The murine monoclonal antibody IgG-1, the murine monoclonal antibody IgG-2, the goat anti-human IgM/RF polyclonal antibody and the rabbit polyclonal antibody IgG are diluted to 10mg/mL, 2mg/mL and 2mg/mL with the blocker buffer solution respectively.
The preparation method of the composite biological blocker for immunochromatography detection comprises the following steps:
s1, preparing a blocker buffer solution, wherein the blocker buffer solution adopts 10mM Tris-HCl buffer solution or 10mM PBS buffer solution, and the pH value of the blocker buffer solution is 7.4+/-0.2;
S2, respectively diluting the murine monoclonal antibody IgG-1, the murine monoclonal antibody IgG-2, the goat anti-human IgM/RF polyclonal antibody and the rabbit polyclonal antibody IgG to 10mg/mL, 2mg/mL and 2mg/mL by using the blocker buffer solution in the step S1;
s3, adding BSA to 10mg/mL of the blocker buffer solution in the step S2;
s4, adding Proclin 300 into the blocker buffer solution in the step S3 to obtain the composite biological blocker.
The fluorescent immunodetection reagent strip comprises a sample pad, a binding pad, a coated nitrocellulose membrane and absorbent paper, wherein the sample pad is treated by a sample pad treatment liquid, and the sample pad treatment liquid comprises a buffer solution with the pH value of 8-10, a surfactant, trehalose, erythrocyte monoclonal antibodies and the composite biological blocker according to any one of claims 1-5, and the composite biological blocker is added into the sample pad treatment liquid according to the volume ratio of 0.5%.
The preparation method of the bonding pad comprises the steps of (1) activating fluorescent microspheres and EDC in phosphate buffer, (2) adding detection antibody I and chicken IgY into the fluorescent microsphere solution for reaction, (3) performing a blocking reaction on the coupled microsphere-antibody complex, and centrifuging to obtain an immunofluorescent microsphere marker, (4) coating the immunofluorescent microsphere marker solution with a prepared concentration on glass fibers, and drying to obtain the bonding pad.
The preparation method of the nitrocellulose membrane comprises the steps of preparing a T line solution by a second detection antibody, preparing a C line solution by sheep anti-chicken IgY, coating the T detection line and the C quality control line area on the nitrocellulose membrane, and drying.
The preparation method of the reagent strip comprises the steps of sequentially adhering a sample pad, a bonding pad, a coated nitrocellulose membrane and absorbent paper to a bottom plate from a sample adding end, and then cutting the sample pad, the bonding pad, the coated nitrocellulose membrane and absorbent paper to required widths to form the test strip.
The composite biological blocker of the invention comprises a plurality of animal immunoglobulins which interfere with a xenotropic antibody, an anti-animal antibody, an anti-rheumatoid factor and the like, has good blocking effect on complex endogenous interfering factors, and can effectively reduce false positive or false negative results of immunochromatography detection caused by endogenous interfering substances.
The performance analysis of the composite bio-blocker reagent is exemplified by immunochromatographic detection of creatine kinase isoenzyme (CKMB):
1) The method is used for the quantitative detection of the fluorescence immunochromatography of the CKBB.
2) 6 Human serum samples (experimental samples, numbers 1-6) with obvious endogenous interference are selected, and 2 human serum samples (control samples, numbers 7-8) with no obvious endogenous interference are selected, the sample pads are treated according to the method, the experimental group is the sample pad added with the blocking agent, the control group is the sample pad not added with the blocking agent, the fluorescent immunodetection test strip is prepared, and the detection experiment is carried out according to the specification, and the results are shown in the following table 1:
TABLE 1
And respectively carrying out correlation analysis on the detection results of the experimental group and the control group and the target value by taking the detection results of the experimental group and the control group as the abscissa and taking the target value as the ordinate, wherein the correlation analysis is shown in fig. 1 and 2.
As can be seen from Table 1, FIG. 1 and FIG. 2, the test strips of the experimental group added with the blocking agent have smaller deviation from the target value and better correlation, while the test strips of the control group without the blocking agent have larger deviation from the target value for the sample detection results of numbers 1-6 and have poor overall correlation. The composite biological blocker can effectively reduce or eliminate the adverse effect of endogenous interfering substances on CKBB fluorescent immunochromatography detection, prevent false positive and false negative detection results, and ensure the accuracy of the detection results.
The foregoing has outlined and described the basic principles, features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the above-described embodiments, and that the above-described embodiments and descriptions are merely illustrative of the principles of the present invention, and that various changes and modifications may be made therein without departing from the spirit and scope of the invention, e.g., forming test strips into plastic card cases, forming reagent cards, which are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. The fluorescent immunodetection reagent strip is characterized by comprising a sample pad, a binding pad, a coated nitrocellulose membrane and absorbent paper, wherein the sample pad is treated by a sample pad treatment liquid, the sample pad treatment liquid comprises a buffer solution with pH value of 8-10, a surfactant, trehalose, erythrocyte monoclonal antibodies and a compound biological blocker, and the compound biological blocker is added into the sample pad treatment liquid according to the volume ratio of 0.5%;
The preparation method of the bonding pad comprises (1) activating fluorescent microsphere and EDC in phosphate buffer solution, (2) adding detection antibody I and chicken IgY into the fluorescent microsphere solution for reaction, (3) performing sealing reaction on coupled microsphere-antibody complex, centrifuging to obtain immunofluorescent microsphere marker, (4) coating the immunofluorescent microsphere marker solution with prepared concentration on glass fiber, and drying to obtain the bonding pad;
the composite biological blocker comprises a blocker buffer solution, wherein the blocker buffer solution is diluted with a murine monoclonal antibody IgG-1, a murine monoclonal antibody IgG-2, a goat anti-human IgM/RF polyclonal antibody and a rabbit polyclonal antibody IgG, and BSA is also added into the blocker buffer solution;
The murine monoclonal antibody IgG-1, the murine monoclonal antibody IgG-2, the goat anti-human IgM/RF polyclonal antibody and the rabbit polyclonal antibody IgG are respectively diluted to 10mg/mL, 2mg/mL and 2mg/mL by the blocker buffer solution;
Wherein the blocker buffer solution is Tris-HCl buffer solution or phosphate PBS buffer solution.
2. The fluoroimmunoassay test strip of claim 1, wherein said blocker buffer is 10mM Tris-HCl buffer or 10mM phosphate PBS buffer.
3. The fluoroimmunoassay test strip of claim 2, wherein the blocker buffer has a pH of 7.4 ± 0.2.
4. The fluorescent immunoassay reagent strip of any one of claims 1-3 wherein the blocker buffer is added to 10mg/mL BSA.
5. The fluoroimmunoassay reagent strip of claim 4, wherein Proclin 300 is further added to said blocker buffer.
6. The preparation method of the composite biological blocker for immunochromatography detection is characterized by comprising the following steps of:
s1, preparing a blocker buffer solution, wherein the blocker buffer solution adopts 10mM Tris-HCl buffer solution or 10mM PBS buffer solution, and the pH value of the blocker buffer solution is 7.4+/-0.2;
S2, respectively diluting the murine monoclonal antibody IgG-1, the murine monoclonal antibody IgG-2, the goat anti-human IgM/RF polyclonal antibody and the rabbit polyclonal antibody IgG to 10mg/mL, 2mg/mL and 2mg/mL by using the blocker buffer solution in the step S1;
s3, adding BSA to 10mg/mL of the blocker buffer solution in the step S2;
s4, adding Proclin 300 into the blocker buffer solution in the step S3 to obtain the composite biological blocker.
7. The fluorescence immunoassay kit according to claim 1, wherein the nitrocellulose membrane is prepared by preparing a T-line solution from a second detection antibody, preparing a C-line solution from a goat anti-chicken IgY, coating the T-line and C-quality control line regions on the nitrocellulose membrane, and drying.
8. The fluoroimmunoassay reagent strip of claim 1, wherein the reagent strip is prepared by the steps of:
The sample pad, the bonding pad, the coated nitrocellulose membrane and the absorbent paper are sequentially adhered on the bottom plate from the sample adding end, and then cut into required widths to form the test strip.
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| CN114895038A (en) * | 2022-04-29 | 2022-08-12 | 江苏浩欧博生物医药股份有限公司 | Enzyme-linked immunoassay method for milk allergen specificity IgE antibody |
| CN115586336A (en) * | 2022-09-14 | 2023-01-10 | 杭州赛基生物科技有限公司 | Kit and preparation method thereof |
| CN115792223A (en) * | 2023-01-13 | 2023-03-14 | 山东康华生物医疗科技股份有限公司 | Anti-cyclic citrullinated peptide antibody detection kit |
| CN116593701B (en) * | 2023-07-19 | 2024-01-23 | 北京豪迈生物工程股份有限公司 | Creatine kinase isoenzyme detection kit containing biological blocker |
| CN117741165A (en) * | 2023-11-29 | 2024-03-22 | 浙江鼎创医疗科技有限公司 | Colloidal gold immunochromatographic test strip for high-specificity and high-sensitivity HNL detection and preparation method and use method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109828119A (en) * | 2019-02-27 | 2019-05-31 | 武汉原谷生物科技有限责任公司 | The mixed type for eliminating the interference of external diagnosis reagent endogenous blocks reagent and its application |
| CN112485433A (en) * | 2020-10-29 | 2021-03-12 | 海卫特(广州)医疗科技有限公司 | Serum amyloid protein A immunochromatographic test strip, kit and preparation method thereof |
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| CN106645681B (en) * | 2016-10-31 | 2018-12-18 | 广州科方生物技术股份有限公司 | A kind of blocking agent kit and its application method for immunochromatographic measurement |
| CN112305231A (en) * | 2020-10-29 | 2021-02-02 | 海卫特(广州)医疗科技有限公司 | N-terminal brain natriuretic peptide precursor immunochromatography test strip, kit and preparation method thereof |
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| CN112485433A (en) * | 2020-10-29 | 2021-03-12 | 海卫特(广州)医疗科技有限公司 | Serum amyloid protein A immunochromatographic test strip, kit and preparation method thereof |
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