Component liquid capable of improving combination efficiency of Biotin and SA magnetic beads and preparation method of component liquid

Abstract

The invention discloses a component liquid capable of improving the combination efficiency of Biotin and SA magnetic beads and a preparation method of the component liquid. The component liquid is mainly composed of a catalyst, other compounds, a surfactant, a metal chelating agent and the like. The component liquid provided by the invention can be applied to the field of detection of chemiluminescence immunodiagnostic reagents, in particular to numerous clinical sample detection items such as nail function, inflammation, hypertension, cytokines, bone metabolism, growth hormone and the like. The method not only can effectively shorten the time required by the reaction process, but also can remarkably amplify the sensitivity of the reagent. The shortening of the reaction time is beneficial to quickly obtaining a detection result in a third-level hospital with a huge sample size, and the method shows remarkable application advantages.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a component liquid capable of improving the binding efficiency between Biotin and SA magnetic beads and a preparation method thereof. Background Art

[0002] Immunodiagnosis is an important sub-sector in the field of in vitro diagnostic reagents. It diagnoses and determines various diseases based on immunological principles, technologies and methods. In diagnostic kits, there are many types of immunodiagnostic reagents, which are widely used in hospitals, blood stations, physical examination centers and other places. In particular, they play a key role in projects such as hepatitis detection, sexually transmitted disease detection, tumor detection and pregnancy detection. Immunodiagnosis includes various types such as radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescence. Its detection method is based on immunological theory and is an experimental process for measuring antigens and antibodies. The core principle is to use the specific binding properties of antigens or antibodies to capture the analyte in the sample, and then use antigens or antibodies bound to luminescent substances to label the analyte, so as to achieve indirect labeling of the analyte with luminescent substances. Finally, the light value of the luminescent substance marked on the analyte is measured by professional instruments, and the concentration of the analyte is calculated by fitting the curve.

[0003] In the application scenario of immunodiagnostic reagents, the specific capture of antigens / antibodies in the sample to be tested by antibodies / antigens is critical. If the capture ability is poor, the antigens / antibodies in trace or low-concentration samples may not be effectively captured or only be captured in small quantities, resulting in an unmeasured concentration or a low measured concentration, resulting in a false-negative result. This is essentially due to insufficient reagent sensitivity. Reagent sensitivity is not only related to the antigen and antibody themselves, but also closely related to the reaction system used by the reagent. A good way to improve sensitivity is to use Biotin combined with SA magnetic beads. The two have extremely strong affinity and can specifically bind to form a stable complex. After the analyte is captured by the specific antibody, another antibody labeled with Biotin binds to it. Because the surface of the SA magnetic beads contains a large amount of SA, the Biotin-labeled antibody can bind to the SA magnetic beads, causing multiple antibodies labeled with luminescent substances to accumulate on the surface of the beads. When the luminescence value is detected, the signal is stronger, which has the effect of amplifying the signal value. However, some projects, such as high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6), have higher requirements for sensitivity and result time (generally within 15 minutes). The required sensitivity cannot be achieved using only the Biotin and SA magnetic bead reaction system. Further optimization is needed to improve the detection sensitivity and binding efficiency to shorten the reaction time. There are many factors that affect the binding efficiency of Biotin and SA magnetic beads, among which the buffer is the most critical. If the buffer system is inappropriate, the binding efficiency of Biotin and SA magnetic beads will be low, and the amount that can be bound per unit time will be much less. Therefore, an adapted buffer system can improve the binding efficiency of Biotin and SA magnetic beads, shorten the reaction time of the project to meet the requirements, and continue to improve the sensitivity of the reagents. In clinical practice applications, it can better meet the requirements.

[0004] Therefore, it is extremely urgent to develop a component solution that can improve the binding efficiency of Biotin and SA magnetic beads. Solving this problem can reduce many difficulties in the development of immunodiagnostic reagents, which is of great significance and can significantly improve various product performance indicators and better meet demand. Summary of the Invention

[0005] The present invention is achieved through the following technical solutions:

[0006] A component solution capable of improving the binding efficiency between Biotin and SA magnetic beads comprises the following components:

[0007] Catalyst: at least one of dibutyltin dilaurate and aluminum isopropoxide;

[0008] Surfactant: at least one of cetyltrimethylammonium bromide and Tween 20;

[0009] Metal chelating agent: at least one of dimercaprol and thioglycolic acid;

[0010] Other compounds: N,N-dimethylformamide, polyvinylpyrrolidone;

[0011] PBS buffer.

[0012] Preferably, the component liquid includes the following components in percentage by mass:

[0013] Catalyst: 0.1-0.5% by mass of dibutyltin dilaurate and 0.1-0.5% by mass of aluminum isopropoxide;

[0014] Surfactant: 0.05-0.15% by mass of cetyltrimethylammonium bromide and 0.05-0.15% by mass of Tween 20;

[0015] Metal chelating agent: 0.05-0.15% dimercaprol and 0.05-0.15% thioglycolic acid;

[0016] Other compounds: 0.5-1.5% by mass of N,N-dimethylformamide and 0.5-1.5% by mass of polyvinylpyrrolidone;

[0017] The balance was 0.1 M PBS buffer.

[0018] Preferably, the pH of the component liquid is 7.4.

[0019] Preferably, the component liquid includes the following components in percentage by mass:

[0020] Catalyst: 0.25% by mass of dibutyltin dilaurate and 0.25% by mass of aluminum isopropoxide;

[0021] Surfactant: 0.1% by mass of cetyltrimethylammonium bromide and 0.1% by mass of Tween 20;

[0022] Metal chelating agent: 0.1% dimercaprol and 0.1% thioglycolic acid;

[0023] Other compounds: 1% by mass of N,N-dimethylformamide and 1% by mass of polyvinylpyrrolidone;

[0024] The balance was 0.1 M PBS buffer.

[0025] Preferably, the component liquid includes the following components in percentage by mass:

[0026] Catalyst: 0.5% by mass of dibutyltin dilaurate and 0.5% by mass of aluminum isopropoxide;

[0027] Surfactant: 0.15% by mass of cetyltrimethylammonium bromide and 0.115% by mass of Tween 20;

[0028] Metal chelating agent: 0.15% by weight of dimercaprol and 0.15% by weight of thioglycolic acid;

[0029] Other compounds: 1.5% by mass of N,N-dimethylformamide and 1.5% by mass of polyvinylpyrrolidone;

[0030] The balance was 0.1 M PBS buffer.

[0031] The present invention also provides a method for preparing a component liquid that can improve the binding efficiency of Biotin and SA magnetic beads, comprising the following steps:

[0032] Add the formulated amount of dibutyltin dilaurate, aluminum isopropoxide, N,N-dimethylformamide, polyvinylpyrrolidone, hexadecyltrimethylammonium bromide, Tween 20, dimercaprol, and thioglycolic acid to 0.1 M PBS buffer, mix well, and adjust the pH value to 7.4.

[0033] The present invention provides a component liquid capable of improving the binding efficiency of Biotin and SA magnetic beads and a preparation method thereof. The component liquid is characterized in that it is composed of a catalyst, other compounds, a surfactant, and a metal chelating agent.

[0034] Catalyst: It is composed of 0.1-0.5% by mass of dibutyltin dilaurate and 0.1-0.5% by mass of aluminum isopropoxide; dibutyltin dilaurate has a certain catalytic activity and can act as a catalyst in the component liquid. It can reduce the activation energy of the binding reaction between Biotin and SA magnetic beads, making the reaction easier to proceed and accelerating the reaction rate, thereby shortening the reaction time to a certain extent and helping to improve the binding efficiency; while aluminum isopropoxide can interact with some hydroxyl groups in the system and regulate the chemical environment of the system. It may promote the specific binding of Biotin and SA magnetic beads by changing the chemical state of the surface of Biotin or SA magnetic beads, making their active sites more easily exposed and interacting, thereby improving the binding efficiency.

[0035] Other compounds: composed of 0.5-1.5% by mass of N,N-dimethylformamide and 0.5-1.5% by mass of polyvinylpyrrolidone; N,N-dimethylformamide is an excellent organic solvent with good solubility and polarity. It can help dissolve other components, making the various substances more evenly distributed in the system, and providing a good homogeneous environment for the binding of Biotin and SA magnetic beads. At the same time, its polarity may help stabilize the transition state during the binding process of Biotin and SA magnetic beads and promote the binding reaction; and polyvinylpyrrolidone has the characteristics of a high molecular polymer. It can form a protective film on the surface of Biotin and SA magnetic beads to prevent them from non-specific aggregation, allowing Biotin and SA magnetic beads to exist in the system in a more dispersed and stable state, increasing their chances of mutual collision and specific binding, thereby improving the binding efficiency.

[0036] Surfactant: Composed of 0.05-0.15% by weight of cetyltrimethylammonium bromide and 0.05-0.15% by weight of Tween 20. Cetyltrimethylammonium bromide is a cationic surfactant that adsorbs onto the surface of SA magnetic beads, altering their surface charge and surface tension. This facilitates electrostatic attraction between the SA beads and the oppositely charged biotin, promoting their binding. It also reduces the surface tension of the solution, facilitating contact and binding between the biotin and SA magnetic beads. Tween 20 is a nonionic surfactant with excellent emulsifying and solubilizing properties. It reduces surface tension at the liquid interface, improving dispersion of the biotin and SA magnetic beads in the solution and preventing aggregation. Furthermore, Tween 20 may also weakly interact with certain groups on the surface of biotin or SA magnetic beads, helping the biotin to better approach and bind to the SA magnetic beads, increasing the probability and efficiency of binding.

[0037] Metal chelator: Composed of 0.05-0.15% by weight of dimercaprol and 0.05-0.15% of thioglycolic acid. Dimercaprol contains a thiol (-SH) group, which has strong nucleophilicity. It may chemically react with active sites on the surface of biotin or SA magnetic beads, forming stable chemical bonds or, through interactions between the thiol group and other groups, linking the biotin and SA magnetic beads, thereby improving their binding efficiency. Thioglycolic acid also contains a thiol group and has similar effects to dimercaprol. Thioglycolic acid can utilize its thiol group to react with specific groups on the surface of biotin or SA magnetic beads, forming covalent bonds or other strong interactions, promoting close binding between biotin and SA magnetic beads and improving binding efficiency. Furthermore, thioglycolic acid may also have a certain effect on the pH of the system, creating an acidic-base environment conducive to the binding reaction.

[0038] The present invention prepares a component liquid that can improve the binding efficiency of Biotin and SA magnetic beads based on actual conditions. Experiments are carried out using the component liquid as a reagent component. Through comparison of experimental results, the component liquid of the present invention can be used in chemiluminescence immunodiagnostic reagents to effectively shorten the reaction time without affecting the measurement results, and the sensitivity is improved to a certain extent. Compared with the measurement results of untreated clinical samples, the measurement results have shortened the time, greatly reduced false negative results, and higher compliance rate.

[0039] The present invention has but is not limited to the following beneficial effects:

[0040] The object of the present invention is to provide a component liquid that can improve the binding efficiency of Biotin and SA magnetic beads. In the reaction process of immunodiagnostic reagents, it is often difficult to improve the binding efficiency of Biotin and SA magnetic beads while shortening the reaction time, and it is also not easy to improve the sensitivity of the product while shortening the reaction time. The present invention successfully overcomes these two major difficulties. First, the reaction time is significantly shortened, so that the experiment can obtain results more quickly, meeting the requirements for detection efficiency; second, the sensitivity of the product is effectively guaranteed, and the missed diagnosis caused by false negatives is effectively avoided, greatly improving the accuracy of the detection. With the help of the component liquid of the present invention, these two difficulties are properly solved, and the sensitivity of the product is further improved while shortening the reaction time. The optimized product has a higher degree of fit with the market demand, thereby effectively enhancing the competitiveness of the product in the market and bringing a more advantageous solution to the field of immunodiagnosis.

[0041] The component liquid that can significantly improve the binding efficiency of Biotin and SA magnetic beads provided by the present invention can be applied in the detection field of chemiluminescence immunodiagnostic reagent, especially in many clinical sample detection projects such as thyroid function, inflammation, hypertension, cytokines, bone metabolism, growth hormone, etc., and the component liquid of the present invention plays a key role. It can not only effectively shorten the time required for the reaction process, but also significantly amplify the sensitivity of the reagent. The shortening of the reaction time is conducive to quickly obtaining test results in tertiary hospitals with huge sample sizes, showing significant application advantages. The improvement of reagent sensitivity is crucial for the accuracy of the actual measured values ​​of clinical samples. Lower sensitivity easily leads to the appearance of false negative results, and then causes the phenomenon of missed diagnosis. In addition, higher reagent sensitivity is usually accompanied by the improvement of luminescence value (RLU) and a wider linear range. The enhancement of sensitivity means that trace concentration samples are more easily accurately detected, effectively avoiding the occurrence of missed diagnosis, and can significantly improve the accuracy of test results. However, the measured value of reagent sensitivity is subject to the comprehensive influence of multiple complex factors, including the performance of the instrument itself, the characteristics of the test item reagents, and the reaction buffer system in the experimental process. Amplifying the sensitivity of reagents while shortening the reaction time, thereby improving the accuracy of measurements and reducing false negative results, has become a very challenging problem in the experimental research and development process. The component liquid that can improve the binding efficiency of Biotin and SA magnetic beads involved in the present invention has successfully overcome this problem, and provides solid and critical technical support for the research and development of immunodiagnostic reagents in clinical testing projects and performance optimization. It is worth mentioning that the component liquid of the present invention, as a key reagent component in the experimental reaction system, can be directly matched with the reagent for application in the instrument detection process, effectively avoiding various interference factors introduced during the manual operation process, greatly improving the reliability and stability of the measurements, and significantly reducing the workload of the experimenters, and comprehensively improving the experimental detection efficiency.

[0042] Specific implementation methods

[0043] The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments are part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results are all within the scope of protection of the present invention.

[0044] Example 1

[0045] Add 0.1% dibutyltin dilaurate, 0.1% aluminum isopropoxide, 0.5% N,N-dimethylformamide, 0.5% polyvinylpyrrolidone, 0.05% hexadecyltrimethylammonium bromide, 0.05% Tween 20, 0.05% dimercaprol, and 0.05% thioglycolic acid to 0.1 M PBS buffer, mix well, and adjust the pH to 7.4.

[0046] Example 2

[0047] Add 0.25% dibutyltin dilaurate, 0.25% aluminum isopropoxide, 1.0% N,N-dimethylformamide, 1.0% polyvinylpyrrolidone, 0.10% hexadecyltrimethylammonium bromide, 0.10% Tween 20, 0.10% dimercaptopropanol, and 0.10% thioglycolic acid to 0.1 M PBS buffer, mix well, and adjust the pH to 7.4.

[0048] Example 3

[0049] Add 0.5% dibutyltin dilaurate, 0.5% aluminum isopropoxide, 1.5% N,N-dimethylformamide, 1.5% polyvinylpyrrolidone, 0.15% hexadecyltrimethylammonium bromide, 0.15% Tween 20, 0.15% dimercaptopropanol, and 0.15% thioglycolic acid to 0.1M PBS buffer, mix well, and adjust the pH to 7.4.

[0050] Example 4

[0051] Add 0.25% aluminum isopropoxide, 1.0% N,N-dimethylformamide, 1.0% polyvinylpyrrolidone, 0.10% hexadecyltrimethylammonium bromide, 0.10% Tween 20, 0.10% dimercaptopropanol, and 0.10% thioglycolic acid to 0.1 M PBS buffer, mix well, and adjust the pH to 7.4.

[0052] Example 5

[0053] Add 0.25% dibutyltin dilaurate, 1.0% N,N-dimethylformamide, 1.0% polyvinylpyrrolidone, 0.10% hexadecyltrimethylammonium bromide, 0.10% Tween 20, 0.10% dimercaptopropanol, and 0.10% thioglycolic acid to 0.1 M PBS buffer, mix well, and adjust the pH to 7.4.

[0054] Data Detection

[0055] The following experimental verification was conducted on two items, high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6), selected from the chemiluminescence immunodiagnostic reagent items. 20 samples with imported reagent test values ​​were collected for each item. The samples were treated with the component solutions prepared in the five examples for 15 minutes and then tested. The specific treatment method was as follows: 90ul of the component solution prepared in the example and 10ul of the sample were added to the reaction cup, incubated at 37°C with shaking for 15 minutes, and then tested on the instrument. Control group 1 was treated with PBS buffer for 15 minutes and then tested, and control group 2 was treated with PBS buffer for 15 minutes and then tested. The test was performed after treatment with PBS buffer for 35 minutes. The specific treatment method of the control group was as follows: 90ul PBS buffer and 10ul sample were added to the reaction cup, among which control group 1 was incubated at 37℃ with shaking for 15min and then tested on the machine, while control group 2 was incubated at 37℃ with shaking for 35min and then tested on the machine. The final results were compared with imported reagents (the imported reagent selected for high-sensitivity C-reactive protein (hs-CRP) was Siemens, and the imported reagent selected for interleukin-6 (IL-6) was Roche), the results were calculated for compliance rate, and then compared respectively.

[0056] Table 1. High-sensitivity C-reactive protein (hs-CRP) test results (cutoff value: <5.0 mg / L)

[0057]

[0058]

[0059]

[0060] Table 2. Interleukin-6 (IL-6) test results (cutoff value: ≤7.0 pg / mL)

[0061]

[0062]

[0063]

[0064]

[0065] From the analysis of the experimental results in the table above, it can be concluded that the results of control group 1 (test results after 15 minutes of treatment with PBS buffer) are generally lower than those of imported reagents, and there are more false negative results. The total compliance rate of high-sensitivity C-reactive protein (hs-CRP) is 70%, and the total compliance rate of interleukin 6 (IL-6) is 75%. It is easy to miss the diagnosis in clinical trials and cause medical disputes. The results of control group 2 (test results after 35 minutes of treatment with PBS buffer) are relatively better. The total compliance rate of high-sensitivity C-reactive protein (hs-CRP) and interleukin 6 (IL-6) is 70%. The rates were 85% and 90% respectively. Although the effect was better, it took a long time, 20 minutes longer than the control group 1, which is not conducive to market promotion and use. The total compliance rate of samples treated with the component liquid of the embodiment of the present invention can reach >90%, and the comparison results are good. The component liquid treatment samples of the three embodiments all took 15 minutes, meeting the client's requirements. Moreover, through analysis, it can be seen that Example 1 has the least amount, and Examples 4 and 5 each lack a catalyst, resulting in false negative results in all three embodiments. The component liquids prepared in Examples 2 and 3 have the best effect. The total compliance rate can reach 100%, without a single false negative result, and the performance is greatly improved. Therefore, the component liquid of the present invention can shorten the reaction time while effectively improving the sensitivity of the immunodiagnostic reagent, ensuring the accuracy of the measured value and avoiding clinical screening and affecting clinical evaluation.

Claims

1. A component solution that can improve the binding efficiency of Biotin and SA magnetic beads, characterized in that Includes the following components: Catalyst: at least one of dibutyltin dilaurate and aluminum isopropoxide; Surfactant: at least one of cetyltrimethylammonium bromide and Tween 20; Metal chelating agent: at least one of dimercaprol and thioglycolic acid; Other compounds: N,N-dimethylformamide, polyvinylpyrrolidone; PBS buffer.

2. The component liquid according to claim 1, characterized in that The composition includes the following components by mass percentage: Catalyst: 0.1-0.5% by mass of dibutyltin dilaurate and 0.1-0.5% by mass of aluminum isopropoxide; Surfactant: 0.05-0.15% by mass of cetyltrimethylammonium bromide and 0.05-0.15% by mass of Tween 20; Metal chelating agent: 0.05-0.15% dimercaprol and 0.05-0.15% thioglycolic acid; Other compounds: 0.5-1.5% by mass of N,N-dimethylformamide and 0.5-1.5% by mass of polyvinylpyrrolidone; The balance was 0.1 M PBS buffer.

3. The component liquid according to claim 2, characterized in that The pH of the component liquid is 7.

4.

4. The component liquid according to claim 2, characterized in that The composition includes the following components by mass percentage: Catalyst: 0.25% by mass of dibutyltin dilaurate and 0.25% by mass of aluminum isopropoxide; Surfactant: 0.1% by mass of cetyltrimethylammonium bromide and 0.1% by mass of Tween 20; Metal chelating agent: 0.1% dimercaprol and 0.1% thioglycolic acid; Other compounds: 1% by mass of N,N-dimethylformamide and 1% by mass of polyvinylpyrrolidone; The balance was 0.1 M PBS buffer.

5. The component liquid according to claim 2, characterized in that The composition includes the following components by mass percentage: Catalyst: 0.5% by mass of dibutyltin dilaurate and 0.5% by mass of aluminum isopropoxide; Surfactant: 0.15% by mass of cetyltrimethylammonium bromide and 0.115% by mass of Tween 20; Metal chelating agent: 0.15% by weight of dimercaprol and 0.15% by weight of thioglycolic acid; Other compounds: 1.5% by mass of N,N-dimethylformamide and 1.5% by mass of polyvinylpyrrolidone; The balance was 0.1 M PBS buffer.

6. A method for preparing a component solution capable of improving the binding efficiency of Biotin and SA magnetic beads according to any one of claims 1 to 5, characterized in that: Add the formulated amount of dibutyltin dilaurate, aluminum isopropoxide, N,N-dimethylformamide, polyvinylpyrrolidone, hexadecyltrimethylammonium bromide, Tween 20, dimercaprol, and thioglycolic acid to 0.1 M PBS buffer, mix well, and adjust the pH value to 7.4.

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