Detection method based on biotin-streptavidin amplification mode, kit and application

Abstract

The invention provides a detection method based on a biotin-streptavidin amplification mode, a kit and application, a biotinylated antibody is prepared by utilizing the characteristic that biotin is combined with the antibody without changing the activity of the biotin, a biotin-streptavidin large compound is formed at a combination pad to amplify a fluorescence signal, and C-line goat anti-rabbit IgG is used for quality control. By screening and optimizing the combination of biotin and streptavidin, complex operation of a conventional mode, such as streptavidin membrane scratching and the like, is avoided. Experiments show that on the premise that the cost is not increased, the NT-proBNP detection sensitivity (which can reach 20 pg / L or even lower) and precision are remarkably improved, the advantages in a low-value section are outstanding, the linearity in a full-concentration range is good, the problem of low-concentration detection in the prior art is effectively solved, a more reliable, efficient, simple and convenient NT-proBNP detection means is provided for clinic, and the application prospect is wide. And diagnosis and disease evaluation of heart failure and other diseases are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis technology, and in particular to a detection method, a kit and an application thereof based on a biotin-streptavidin amplification mode. Background Art

[0002] NT-proBNP is an inactive N-terminal polypeptide fragment produced by endonuclease cleavage of pro-brain natriuretic peptide (pro-BNP). It is mainly present in the secretions produced by myocardial cells in response to ventricular wall tension or local ischemia. A large number of studies have shown that NT-proBNP has a long half-life and strong stability. It can be used as the main indicator for the diagnosis of heart failure and plays an important role in the diagnosis and prognosis of the disease. Currently, the main methods for quantitative detection of NT-proBNP are: radioimmunoassay technology, enzyme-linked immunosorbent assay, electrochemiluminescence immunoassay and fluorescent immunochromatography technology. Radioimmunoassay technology has a low cost, but there is a potential for radioactive contamination, and the reagents have a half-life, and are gradually replaced by non-radiolabeled immunoassay technology. Enzyme-linked immunosorbent assay is cumbersome, time-consuming and labor-intensive, and has low accuracy and sensitivity. Electrochemiluminescence immunoassay is widely used for quantitative detection of NT-proBNP, among which Roche Elecsys electrochemiluminescence immunoassay system is the most representative. However, this analytical method requires a large number of samples to reduce costs and is not suitable for quantitative and rapid detection of NT-proBNP in small and medium-sized institutions and individuals. Immunochromatography is an immunoassay technology based on chromatography and antigen-antibody specific binding that emerged in the 1990s. At present, immunochromatography technology has developed into many different types, and the most commonly used is the double antibody / antigen sandwich method. In the NT-proBNP immunochromatography test, since the T-line antibody and antigen-fluorescein complex have only an action time of 15s, the effective detection limit of NT-proBNP needs to be 20pg / mL. Under the conditions of low antigen concentration and extremely short reaction time, it is difficult for a single immunochromatography technology to achieve accurate detection of low-concentration NT-proBNP. Although biotin and streptavidin have highly specific affinity and multi-stage amplification effects, traditional application methods still have limitations.

[0003] Therefore, in view of the above problems, the present invention urgently needs to find a NT-proBNP detection method, kit and application based on the biotin-streptavidin amplification mode. By carefully designing the binding mode of biotin and streptavidin, an efficient amplification system is constructed at the binding pad, which overcomes many disadvantages of the prior art and achieves a comprehensive improvement in detection performance. The sensitivity of immune binding and tracer analysis can be greatly improved, and a method combining the biotin-streptavidin system with fluorescent immunochromatography technology is adopted to achieve simple, rapid and sensitive detection of NT-proBNP. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a detection method, a kit and an application based on a biotin-streptavidin amplification mode. Through the specific binding of biotin and streptavidin and the multi-stage amplification effect, in the test strip detection system, the NT-proBNP coated antibody is fixed on the NC membrane. After the antigen in the sample binds to it, the pre-mixed biotinylated labeled antibody and the streptavidin-fluorescein complex at the binding pad play a key role to form an immune complex containing fluorescein, and the antigen content is determined by detecting the fluorescent signal. At the same time, the amplification effect is improved by screening and optimizing the types of biotin and streptavidin and modifying streptavidin. The present invention is different from the conventional biotin-streptavidin amplification mode. It innovatively pre-mixes the relevant complexes at the binding pad, avoiding complicated operations such as streptavidin stripping, simplifying the process and reducing errors, significantly improving the sensitivity (up to 20pg / L or even lower) and precision of low-concentration NT-proBNP detection, and effectively solving the shortcomings of the prior art in low-concentration detection, providing a more reliable and efficient detection method for clinical diagnosis, and playing an important role in the field of NT-proBNP detection.

[0005] In order to achieve the above object, the present invention adopts the following scheme:

[0006] On the one hand, the present invention provides a detection kit based on a biotin-streptavidin amplification mode, comprising a test strip, wherein the test strip comprises a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad, wherein a detection line and a quality control line are provided on the nitrocellulose membrane, the conjugation pad comprises a labeled antibody, streptavidin, fluorescein and biotin, the detection line comprises a coated antibody, and the quality control line is coated with goat anti-rabbit IgG.

[0007] In the existing technology, the detection of NT-proBNP faces many challenges. Although the combination of biotin and streptavidin has the potential to amplify the detection signal, the traditional application method still has obvious shortcomings in achieving high sensitivity and high accuracy detection of low-concentration NT-proBNP. For example, when detecting NT-proBNP with immunochromatography technology, due to the extremely short action time of T-line antibodies and antigen-fluorescein complexes, it is difficult for conventional methods to achieve accurate detection of low-concentration NT-proBNP, and the effective detection limit is difficult to meet clinical needs. At the same time, although some existing detection methods have attempted to use the biotin-streptavidin amplification system, the operation is complicated, such as the need to accurately control the amount of streptavidin membrane and the step-by-step preparation and addition of complex biotinylated antibody complexes, and the detection sensitivity and precision are limited. It is easily affected by fluctuations in experimental conditions, resulting in poor stability of the test results, and cannot provide reliable low-concentration NT-proBNP test results for the clinic, which is not conducive to the early diagnosis and disease monitoring of diseases such as heart failure.

[0008] In order to overcome the above problems, the present invention has innovatively designed a new detection mode based on biotin-streptavidin synergistic amplification. By pre-mixing the biotinylated NT-proBNP labeled antibody and the streptavidin-coupled fluorescein complex at the binding pad, and then utilizing the highly specific affinity and multi-stage amplification effect between biotin and streptavidin, when the NT-proBNP antigen in the sample binds to the coated antibody on the nitrocellulose membrane, the complex at the binding pad reacts with it to form an immune complex containing fluorescein, thereby obtaining an efficient fluorescence signal amplification effect. Compared with the traditional biotin-streptavidin amplification mode and conventional detection methods, the design of the present invention has a more significant effect. In terms of sensitivity, it can detect NT-proBNP as low as 20pg / L or even lower concentrations, with obvious advantages in the low-value segment, effectively solving the problem of low-concentration detection in existing technologies; in terms of precision, the coefficient of variation (CV) of detection of samples with different concentration gradients is significantly reduced, ensuring the accuracy of the test results; in terms of operational procedures, it simplifies experimental operations, avoids complex steps such as streptavidin stripping, reduces the requirements for experimental personnel's operating skills, improves experimental efficiency and stability, and is more suitable for clinical rapid detection scenarios, providing clinical diagnosis with a more reliable, efficient and simple NT-proBNP detection method, which is helpful for the early diagnosis and condition assessment of diseases such as heart failure.

[0009] Furthermore, the labeled antibody is labeled with biotin to form a biotinylated labeled antibody, and the streptavidin is coupled with fluorescein to form streptavidin-fluorescein.

[0010] Furthermore, the biotinylated labeled antibody specifically recognizes the antigen that has been bound to the coated antibody to form an immune complex of "coated antibody-antigen-biotinylated labeled antibody".

[0011] Furthermore, the streptavidin-fluorescein binds to the biotin on the biotinylated labeled antibody, and introduces the fluorescein into the immune complex, thereby achieving multiple amplification of the fluorescence signal.

[0012] Furthermore, in the test strip, streptavidin-conjugated fluorescein and biotinylated labeled antibody are first fixed to the conjugate pad, and then the detection line and quality control line are respectively drawn on the nitrocellulose membrane, and finally the sample pad, conjugate pad, nitrocellulose membrane, and absorption pad are overlapped on the bottom plate in sequence.

[0013] Furthermore, the biotin includes any one or more combinations of EZ-Link Sulfo-NHS-LC-Biotin, EZ-Link Sulfo-NHS-LC-LC-Biotin, and EZ-Link NHS-PEG12-Biotin.

[0014] In the NT-proBNP detection process, biotin is a key component in the biotin-streptavidin amplification system. Its binding effect with the NT-proBNP labeled antibody directly affects the performance of the entire amplification system, and thus determines the accuracy and sensitivity of the detection results. However, biotin with different structures and characteristics may have significant differences in binding with antibodies and in the subsequent signal amplification process. However, in the existing technology, there is a lack of systematic and in-depth research and optimization screening for biotin types suitable for NT-proBNP detection.

[0015] Therefore, in some embodiments, in order to further optimize the detection performance of the biotin amplification system, different biotin types connected to the NT-proBNP labeled antibody were compared and screened, and the experimental results showed that different biotin types had a significant impact on the effect of the biotin amplification system of the present invention in detecting NT-proBNP. EZ-Link NHS-PEG12-Biotin showed the best comprehensive performance among the biotins tested in this experiment, and had obvious advantages in terms of improving the detection sensitivity at low concentrations and ensuring the accuracy and stability of the detection at high concentrations. This also further verified that our idea of ​​optimizing the detection system by screening different biotin types is correct and necessary, providing strong support for the realization of high-sensitivity and high-accuracy NT-proBNP detection, helping to improve the reliability and effectiveness of clinical diagnosis, and also providing valuable data and reference for subsequent in-depth research on the application of the relationship between biotin structure and function in immunoassays.

[0016] Similarly, streptavidin plays an important role in signal transduction and amplification in the biotin-streptavidin amplification mode, and the properties of the complex labeled with fluorescein are directly related to the accuracy and sensitivity of the test results. However, streptavidin of different sources, properties and structures may behave very differently in the process of binding to fluorescein and interacting with biotinylated antibodies. Previously, the most suitable type of streptavidin in the NT-proBNP detection system has not been fully explored.

[0017] Therefore, in some embodiments, in order to further optimize the detection performance of the biotin amplification system, different types of streptavidin connected to fluorescein were also compared and screened. The experimental results show that different types of streptavidin have significant and diverse effects on the effect of the biotin amplification system of the present invention in detecting NT-proBNP. The preferred streptavidin 5 of the present invention shows extremely outstanding comprehensive performance in both low-concentration and high-concentration sample detection, and is significantly better than other streptavidin in terms of T / C value and stability. This is because in the low-concentration NT-proBNP detection scenario, the source and nature of streptavidin have a crucial influence on its binding ability with biotinylated antibodies and fluorescein, as well as the subsequent signal amplification effect. Streptavidin produced by different manufacturers may differ in purity, activity, compatibility with other reagents, and spatial structure, which leads to different performance in the detection system. Among them, the single-site or double-site streptavidin can bind more fluorescein after modification. In the present invention, streptavidin 5 is a single binding site. Compared with the conventional four-binding site streptavidin, when binding with the biotinylated antibody and fluorescein, there is no competition caused by too many sites, which leads to reduced sensitivity. In the low-concentration NT-proBNP detection scenario, this structural characteristic enables streptavidin 5 to bind to the biotin on the biotinylated antibody more accurately, avoiding the problem of reduced binding efficiency caused by site competition, thereby effectively enhancing the binding ability with the biotinylated antibody, and laying a good foundation for subsequent signal amplification. This fully proves the importance and value of our screening and optimization of streptavidin types. The preferred streptavidin can significantly optimize the detection effect, effectively realize high-sensitivity and high-accuracy NT-proBNP detection, and provide solid and reliable technical support for clinical diagnosis. At the same time, it also provides a scientific and accurate reference basis for the subsequent application of streptavidin in this detection system, which is helpful to promote the further development and improvement of detection technology in this field.

[0018] Furthermore, the coated antibody includes NT-proBNP coated antibody, and the labeled antibody includes NT-proBNP labeled antibody.

[0019] Furthermore, the concentration of the coated antibody is 1-3 mg / mL.

[0020] Furthermore, the molar ratio of the labeled antibody to biotin is 1:10-1:30.

[0021] Furthermore, the molar ratio of the fluorescent streptomycin to biotin is 4:1-1:1.

[0022] In some embodiments, the molar ratio of fluorescent streptavidin to biotin is also optimized and screened within the range of 4:1-1:1. The experiment uses the biotin-streptavidin amplification mode of the present invention to prepare a test strip for detecting NT-proBNP, and conducts detection for NT-proBNP standards of different concentrations. The results show that within this molar ratio range, good detection effects are exhibited. This shows that within the molar ratio range of 4:1-1:1, different molar ratios have their own advantages in different concentration ranges, and can provide good detection performance for the detection method of the present invention as a whole, ensuring that relatively accurate and sensitive NT-proBNP detection is achieved within the full concentration range (20pg / L-35000pg / L), providing reliable support for clinical diagnosis, and also providing valuable reference data for related research in this field.

[0023] In another aspect, the present invention provides a method for detecting NT-proBNP, wherein the kit as described in any one of the above technical solutions is used to detect NT-proBNP.

[0024] Furthermore, the detection method comprises the following steps:

[0025] (1) Add the sample to be tested to the sample pad and allow the sample to migrate on the test strip;

[0026] (2) The NT-proBNP antigen in the sample binds to the NT-proBNP monoclonal antibody on the nitrocellulose membrane and reacts with the biotinylated NT-proBNP labeled antibody and streptavidin-conjugated fluorescein complex on the binding pad to form an immune complex;

[0027] (3) Detect the fluorescence signal intensity on the immune complex and determine the NT-proBNP content in the sample based on the standard curve.

[0028] In the process of developing the NT-proBNP detection method of the present invention, the present invention is committed to exploring a more efficient, accurate and clinically applicable detection scheme. Although there are many methods for detecting NT-proBNP, they still have defects to varying degrees in terms of sensitivity, precision, ease of operation, etc. In order to further verify the advantages of the detection method based on the biotin-streptavidin amplification mode designed by the present invention, in some embodiments, three different amplification modes, namely the conventional double antibody sandwich mode, the conventional streptavidin amplification mode and the optimized biotin amplification mode of the present invention, are used to prepare test strips for detecting NT-proBNP under the same conditions, and the test results are compared and analyzed.

[0029] The experimental results show that in terms of sensitivity comparison, in the low concentration range (such as 20pg / L-1000pg / L), the optimized biotin amplification mode of the present invention has a significantly higher detection signal intensity than the conventional double antibody sandwich mode and the conventional streptavidin amplification mode. For example, at a concentration of 20pg / L, the detection result of the double antibody sandwich mode is 429, the ordinary amplification mode is 1000, and the innovative amplification mode of the present invention reaches 1133. As the concentration decreases, this advantage becomes more obvious. This shows that the mode of the present invention can effectively improve the detection sensitivity of low-concentration NT-proBNP, and can detect as low as 20pg / L, which meets the clinical needs for low-concentration sample detection, while other modes have relatively weak detection capabilities in the low-concentration range.

[0030] In the linear range and amplification effect evaluation within the entire concentration gradient range (20pg / L-35000pg / L), the biotin amplification mode detection results of the present invention show a good linear relationship, and the signal intensity increases significantly. Compared with the conventional double antibody sandwich mode, the detection signal of the present invention in the medium and high concentration range (such as 1000pg / L-35000pg / L) is significantly improved. For example, at a concentration of 35000pg / L, the double antibody sandwich mode detection value is 26966, while the improved amplification mode of the present invention reaches 73792. Compared with the conventional streptavidin amplification mode, the present invention also has obvious advantages at each concentration point, indicating that the amplification effect of the present invention is more significant, and can more accurately reflect the concentration changes of NT-proBNP in the sample, effectively expanding the linear range of detection, and improving the accuracy and reliability of detection.

[0031] In addition, compared with the conventional streptavidin amplification mode, the present invention reduces complex operations such as streptavidin membrane streaking in terms of operating steps, simplifies the experimental process, improves experimental efficiency, and is more conducive to application in clinical rapid detection scenarios.

[0032] In summary, these experimental results fully demonstrate that the detection scheme based on biotin-streptavidin synergistic amplification proposed in the present invention has better effects such as high sensitivity, wide linear range, and simple operation, and can effectively solve many problems existing in the prior art in the detection of NT-proBNP, and provides a more efficient and accurate detection method for clinical diagnosis, which has important application value and promotion prospects.

[0033] On the other hand, the present invention provides a use of an immune complex for preparing a reagent for improving the sensitivity and accuracy of antigen detection, wherein the immune complex includes an immune complex of "coated antibody-antigen-biotinylated labeled antibody", and fluorescein is introduced into the immune complex by binding streptavidin-fluorescein to biotin on the biotinylated labeled antibody, thereby achieving multiple amplification of the fluorescence signal.

[0034] Furthermore, the kit is as described in any one of the above technical solutions.

[0035] The beneficial effects of the present invention are:

[0036] 1. The present invention is based on the biotin-streptavidin amplification mode. By carefully designing the combination mode of biotin and streptavidin in the test strip detection system, such as pre-mixing the biotinylated labeled antibody and the streptavidin-fluorescein complex at the conjugate pad, low-concentration NT-proBNP can be effectively captured. In the experiment, for NT-proBNP samples as low as 20pg / L, a fluorescent signal significantly stronger than the conventional detection mode can still be generated. Compared with the conventional double antibody sandwich mode and the conventional streptavidin amplification mode in the prior art, the detection signal intensity is significantly improved in the low concentration range (20pg / L-1000pg / L), and extremely low concentrations of NT-proBNP can be accurately detected, meeting the clinical needs for low-concentration sample detection in early disease diagnosis, and providing strong support for early detection and intervention of diseases. At the same time, it not only performs well in the low value segment, but also shows a good linear relationship in the entire detection concentration range (20pg / L-35000pg / L), and the signal intensity growth rate is reasonable. Compared with conventional detection modes, it can more accurately reflect the concentration changes of NT-proBNP in samples in the medium and high concentration ranges (1000pg / L-35000pg / L), effectively avoiding detection errors caused by insufficient detection sensitivity or signal saturation, greatly improving the precision of detection, ensuring the accuracy of clinical test results, and helping in clinical disease assessment and formulation of treatment plans.

[0037] 2. Unlike the traditional biotin-streptavidin amplification mode, the present invention innovatively makes key improvements at the binding pad, avoiding tedious steps such as precise control of the amount of streptavidin membrane and the step-by-step preparation and addition of complex biotinylated antibody complexes. By pre-mixing the relevant complexes, the experimental operation process is simplified, the requirements for the experimental personnel's operating skills are reduced, the experimental errors caused by complex operations are reduced, and the repeatability and stability of the experiment are improved. This simplified operation procedure makes the detection process more convenient and faster, and is more suitable for clinical field testing or small and medium-sized medical institutions and other scenarios with high requirements for detection efficiency and ease of operation. There is no need for complex equipment and professional technicians to operate for a long time, which helps to increase the widespread application of NT-proBNP testing in clinical practice and promote the early diagnosis and timely treatment of related diseases such as heart failure.

[0038] 3. The present invention specifically screens a variety of biotin types and streptavidin types, and modifies streptavidin (such as optimizing its coupling conditions and purity with fluorescein), and finds the best synergistic amplification combination. When the biotin with modified arm length is combined with the streptavidin with optimized single binding site and high binding site with fluorescein, a more stable complex with strong signal amplification ability can be formed at the binding pad. This optimized combination makes the interaction between biotin and streptavidin more efficient, which can promote more fluorescein to be labeled on the immune complex, significantly enhances the intensity of the fluorescent signal, and further improves the overall amplification effect of the biotin amplification system. The stronger amplification effect enables the present invention to detect NT-proBNP more sensitively and accurately, meeting the growing clinical demand for NT-proBNP detection. In the diagnosis and monitoring of diseases such as heart failure, more accurate NT-proBNP detection helps doctors better judge the severity of the disease, evaluate the treatment effect and predict the progression of the disease, providing a more reliable basis for clinical decision-making, and improving the quality of medical care and the treatment effect of patients.

[0039] 4. The present invention sets sheep anti-rabbit IgG at the C line (control line) of the test strip, which can intercept the independent system rabbit IgG fluorescent antibody. By observing the fluorescent signal at the C line, it is possible to quickly and intuitively judge whether the detection process is proceeding normally, such as whether the sample is added correctly, whether the test strip is valid, etc. If the expected fluorescent signal or signal abnormality does not appear at the C line, it indicates that there may be problems in the detection process, and it is necessary to retest or check the test strip and the detection conditions, thereby effectively ensuring the reliability and accuracy of the test results. This quality control mechanism provides additional quality control guarantees for the entire detection process, and enhances the confidence of clinicians and patients in the test results. In clinical diagnosis, reliable test results are crucial for the accurate diagnosis and rational treatment of diseases. The quality control design of the present invention helps to reduce the occurrence of false positive and false negative results and improve the clinical application value of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the process of detecting NT-proBNP using the existing biotin amplification system.

[0041] Figure 2 This is a process of detecting NT-proBNP using the biotin-streptavidin synergistic amplification mode of the present invention.

[0042] Figure 3 This is a process of forming a complex using a conventional double antibody sandwich model.

[0043] Figure 4 The process of complex formation using conventional streptavidin amplification mode.

[0044] Figure 5 The invention relates to a process of forming a complex by using the optimized biotin amplification mode of the present invention. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not have any limiting effect on it. Unless otherwise specified, the reagents used in the examples of the present invention are all commercially available.

[0046] Example 1 Optimal preparation method of the test strip for detecting NT-proBNP of the present invention

[0047] In the preliminary experiments, the present invention used the existing biotin amplification system to detect NT-proBNP. The specific detection process is as follows: Figure 1 As shown, the principle of the existing conventional double antibody sandwich chromatography is adopted. The results show that although there is a certain amplification effect, the detection sensitivity and precision of extremely low concentration NT-proBNP are not significantly improved. Therefore, in the prior art, the conventional streptavidin amplification mode is adopted: SA (streptavidin) is used for membrane stripping, NT-proBNP coated antibody and biological coupling form a coated antibody-biotin complex, and NT-proBNP labeled antibody is labeled with fluorescein to form a labeled antibody-fluorescein complex. Although the principle has certain advantages over the conventional double antibody sandwich chromatography and improves the biotin amplification effect, the method has the disadvantages of complex operation and the sensitivity of low concentration NT-proBNP detection is still not ideal. For example, the amount of streptavidin stripping and the preparation process of the biotinylated antibody complex need to be precisely controlled, which is easily affected by fluctuations in experimental conditions, resulting in poor stability of the detection results.

[0048] For example, in the invention patent with the authorization announcement number CN113092790B and the invention name: A detection kit for procalcitonin and its preparation method, two microspheres with different particle sizes, two PCT monoclonal antibodies and one PCT polyclonal antibody are used. The large-diameter microspheres are modified with streptavidin, and the small-diameter microspheres are not treated. The streptavidin and biotin amplification system is used to connect the large-diameter microspheres with the streptavidin, and the polyclonal antibody is labeled with biotin to form a large latex microsphere-streptavidin-biotin-polyclonal antibody amplification system, thereby improving its sensitivity at the low value end, and at the same time, the two monoclonal antibodies are labeled with small-diameter microspheres to improve their linearity. The conventional streptavidin amplification mode adopted in the patent can improve the sensitivity at the low value end to a certain extent, but its operation is complicated, involving multi-step labeling and mixing and special equipment and reagent requirements; at the same time, the improvement of low-concentration detection sensitivity is limited and is greatly affected by fluctuations in experimental conditions; the linear range is not fully optimized, and the accuracy in the high-concentration area is reduced; and the stability of the complex is poor, which affects the accuracy and reliability of the detection results, and cannot meet the needs such as the precise detection of NT-proBNP of the present invention.

[0049] Therefore, in order to further significantly improve the amplification effect and achieve more accurate and sensitive detection of NT-proBNP, the present invention proposes an innovative biotin-streptavidin synergistic amplification mode, the principle of which is as follows (specifically Figure 2As shown in the figure): In the test strip detection system, the NT-proBNP coated antibody is first fixed on the nitrocellulose membrane (NC membrane) as the capture antibody. When the NT-proBNP antigen in the sample binds to the capture antibody, the pre-mixed biotinylated NT-proBNP labeled antibody and streptavidin-coupled fluorescein complex play a key role at the binding pad. The biotinylated labeled antibody can specifically recognize and bind to the antigen that has bound to the capture antibody to form an immune complex of "coated antibody-antigen-biotinylated labeled antibody"; at the same time, streptavidin and biotin have high specificity and strong affinity. Streptavidin-coupled fluorescein can introduce fluorescein into the immune complex by binding to the biotin on the biotinylated labeled antibody, thereby achieving efficient amplification of the fluorescent signal. At the same time, by specifically screening a variety of biotin species and streptavidin species, and modifying streptavidin (optimizing its coupling conditions and purity with fluorescein), the best synergistic amplification combination is explored. After a large number of experiments, it was found that when a specific biotin and optimized streptavidin are used in combination, the two can more efficiently form a stable complex with strong signal amplification capabilities at the binding pad. The specific binding of biotin and streptavidin causes more fluorescein to be labeled on the immune complex, greatly enhancing the intensity of the fluorescent signal, thereby effectively improving the detection sensitivity of NT-proBNP. At the same time, this synergistic amplification mode simplifies the operation process, avoids complex and easily interfered steps such as traditional streptavidin membrane, reduces experimental errors, and improves the stability and accuracy of the test results. It shows excellent performance in the entire detection concentration range (especially the low value segment), and can provide a more reliable and sensitive detection method for NT-proBNP detection, meeting the needs of clinical diagnosis for accurate detection of low-concentration samples.

[0050] The present invention provides an optimal preparation method for a test strip for detecting NT-proBNP, comprising the following steps:

[0051] 1. Immobilize streptavidin-conjugated fluorescein and biotinylated NT-proBNP monoclonal antibody to the binding pad

[0052] 1. Preparation of Fluorescein-labeled Streptavidin

[0053] Pipette 5 μL of fluorescein and add 0.5 mg of streptavidin, add appropriate amount of NaHCO3 and dilute to a total volume of 200 μL with PB buffer, incubate for 3 hours with rotation; after the incubation, add 10% BSA to block for 1 hour; finally, pass through a chromatography column to collect the labeled streptavidin-fluorescein for later use.

[0054] 2. Preparation of biotinylated NT-proBNP monoclonal antibody 1

[0055] Take 0.2 mg of antibody, add NHS-PEG12-Biotin at a molar ratio of 1:20 to antibody / activated biotin, with a total volume of 100 μL, and incubate at room temperature for 30 min. Dilute to 5 times the volume of the reaction solution with PB buffer, dialyze at 4°C for 72 h, and take out for use.

[0056] 3. Preparation of conjugate pad

[0057] The conjugate pad was treated by spraying 0.3 mL of 25 mM Tris containing 1% Tween-20 and 0.5% Casein at 10 μL / cm for 30 min. The treated conjugate pad was taken out and dried at 37°C for 12 hours. After drying, the previously prepared fluorescein-streptavidin and biotin NT-proBNP monoclonal antibody 1 (labeled antibody) were sprayed on the conjugate pad respectively, dried at 37°C for 12 hours, and set aside.

[0058] 2. Draw T line NT-proBNP monoclonal antibody and C line goat anti-rabbit IgG on the nitrocellulose membrane

[0059] 4. Preparation of nitrocellulose membrane (NC membrane)

[0060] NT-proBNP monoclonal antibody 2 (coating antibody) and goat anti-rabbit IgG were added to 0.01 mol / L PBS containing 2% trehalose to prepare 2 mg / mL streptavidin stripping solution and 0.5 mg / mL goat anti-rabbit IgG stripping solution.

[0061] Use a film sprayer to streak a 2 mg / mL NT-proBNP monoclonal antibody 2 streaking solution on the nitrocellulose membrane as the T line of the test strip; streak a 0.5 mg / mL goat anti-rabbit IgG streaking solution on the nitrocellulose membrane as the C line of the test strip. Place the treated and streaked nitrocellulose membrane at 37°C to dry for 24 hours for later use.

[0062] 3. Assembly and cutting of test strips

[0063] 5. The sample pad, the prepared binding pad, the dried nitrocellulose membrane (NC membrane), and the absorption pad are overlapped and pasted on the PVC bottom plate in sequence, and then cut into test strips of appropriate size (about 4 mm in the present invention).

[0064] The present invention provides a method for improving the performance of solid-phase NT-proBNP chromatography detection by using a biotin amplification system, proposes an innovative principle based on the synergistic effect of biotin-streptavidin, and makes key improvements at the conjugate pad to prepare the best test strip for detecting NT-proBNP, couples the NT-proBNP labeled antibody with biotin to form a labeled antibody-biotin complex, and simultaneously labels fluorescein with streptavidin at a specific ratio (such as antibody: fluorescein = 1 mg: 10 μL) to form a streptavidin-fluorescein complex, and the two are pre-mixed and sprayed on the conjugate pad at a spray volume of 4 μL / cm, dried at 37°C overnight, and then assembled with other components into a test strip. This design allows biotin and streptavidin to form a stable large complex in advance at the conjugate pad, laying a solid foundation for subsequent binding with the antigen and amplification of the fluorescence signal. At the same time, the new streptavidin amplification mode of the present invention has achieved significant improvement compared with the traditional mode and the conventional streptavidin amplification mode. The detection sensitivity of the present invention for NT-proBNP can reach 20pg / L or even lower, and the detection precision in the low value segment is also significantly improved, such as the coefficient of variation (CV) of the detection of samples with different concentration gradients is significantly reduced, ensuring the accuracy and reliability of the detection results, effectively solving the shortcomings of the existing technology in the detection of low-concentration NT-proBNP, and providing more powerful support for clinical diagnosis. At the same time, the present invention optimizes the operating steps, reduces the complex operations in the experimental process, improves the experimental efficiency, and is more suitable for the needs of clinical rapid detection.

[0065] Example 2 Comparative study of the detection effects of different amplification modes on NT-proBNP

[0066] To further illustrate the significant effect and advantages of the biotin amplification system proposed in the present invention in the detection of NT-proBNP, this example uses three different amplification modes, namely the conventional double antibody sandwich mode, the conventional streptavidin amplification mode and the optimized biotin amplification mode of the present invention, to prepare test strips for detecting NT-proBNP under the same conditions, and compare and analyze the test results. The specific process is as follows:

[0067] 1. Conventional double antibody sandwich model (specifically Figure 3 (shown)

[0068] 1) NT-proBNP-coated antibody was applied to the NC membrane at a spray volume of "2 mg / mL, 1 μL / cm" and dried at 37°C overnight;

[0069] 2) Label the NT-proBNP labeled antibody at a ratio of "antibody: fluorescein = 1 mg: 10 μL", spray it on the conjugate pad at a spray volume of 4 μL / cm, and dry it at 37°C overnight;

[0070] 3) Combine the membrane and conjugate pad to form a slab test gradient sample.

[0071] 2. Conventional streptavidin amplification mode (specifically Figure 4 (shown)

[0072] 1) Streptavidin was applied to the NC membrane at a rate of 2 mg / mL, 1 μL / cm and dried at 37°C overnight;

[0073] 2) coupling the NT-proBNP coated antibody to biotin to form a coated antibody-biotin complex;

[0074] 3) labeling the NT-proBNP labeled antibody at a ratio of "antibody:fluorescein = 1 mg:10 μL" to form a labeled antibody-fluorescein complex;

[0075] 4) Spray the coated antibody-biotin complex and the labeled antibody-fluorescein complex onto the conjugate pad at a spray volume of 4 μL / cm and dry at 37°C overnight;

[0076] 5) Combine the membrane and conjugate pad to form a slab test gradient sample.

[0077] 3. The optimized biotin amplification mode of the present invention (specifically as Figure 5 (shown)

[0078] 1) NT-proBNP-coated antibody was applied to the NC membrane at a spray volume of "2 mg / mL, 1 μL / cm" and dried at 37°C overnight;

[0079] 2) coupling the NT-proBNP labeled antibody to biotin to form a labeled antibody-biotin complex;

[0080] 3) Labeling streptavidin at a ratio of "antibody:fluorescein = 1 mg: 10 μL" to form a streptavidin-fluorescein complex;

[0081] 4) Premix the labeled antibody-biotin complex and the streptavidin-fluorescein complex to form a fluorescein-amplified complex, spray the complex on the conjugate pad at a spray volume of 4 μL / cm, and dry at 37°C overnight;

[0082] 5) Combine the membrane and conjugate pad to form a slab test gradient sample.

[0083] By testing the test strips prepared by three different amplification modes, the NT-proBNP test results under different concentration gradients were obtained. The specific test results are shown in Table 1:

[0084] Table 1. Comparison of NT-proBNP test results in different amplification modes

[0085] Concentration (pg / L) Double Antibody Sandwich Normal zoom Improved Amplification Test 1 20 429 1000 1133 Test 2 100 682 1155 1399 Test 3 250 853 1743 2441 Test 4 500 936 1920 2753 Test 5 1000 1436 2953 3650 Test 6 2000 2151 4418 5837 Test 7 4375 4384 8621 11307 Test 8 8750 9360 18466 24538 Test 9 17500 19450 36337 54323 Test 10 35000 26966 56116 73792

[0086] 1. Sensitivity comparison

[0087] It can be clearly seen from the data in Table 1 that within the low concentration range (such as 20pg / L-1000pg / L), the optimized biotin amplification mode of the present invention has a significantly higher detection signal intensity than the conventional double antibody sandwich mode and the conventional streptavidin amplification mode. For example, at a concentration of 20pg / L, the detection result of the double antibody sandwich mode is 429, the ordinary amplification mode is 1000, and the innovative amplification mode of the present invention reaches 1133. As the concentration decreases, this advantage becomes more obvious. This shows that the mode of the present invention can effectively improve the detection sensitivity of low-concentration NT-proBNP, and can detect as low as 20pg / L, meeting the clinical demand for low-concentration sample detection, while other modes have relatively weak detection capabilities in the low concentration range. This may be because the modified streptavidin is coupled with more fluorescent substances, and then combined with the labeled antibody through biotin, so that the overall luminescence efficiency is stronger than directly coupling the labeled antibody with the fluorescent substance. In conventional streptavidin membranes, due to steric hindrance, the number of biotins that streptavidin can bind to is reduced.

[0088] 2. Linear range and amplification effect evaluation

[0089] In the entire concentration gradient range (20pg / L-35000pg / L), the detection results of the biotin amplification mode of the present invention show a good linear relationship, and the signal intensity increases greatly. Compared with the conventional double antibody sandwich mode, the detection signal of the present invention in the medium and high concentration range (such as 1000pg / L-35000pg / L) is significantly improved. For example, at a concentration of 35000pg / L, the detection value of the double antibody sandwich mode is 26966, while the improved amplification mode of the present invention reaches 73792. Compared with the conventional streptavidin amplification mode, the present invention also has obvious advantages at each concentration point, indicating that the amplification effect of the present invention is more significant, and can more accurately reflect the concentration changes of NT-proBNP in the sample, effectively expanding the linear range of detection, and improving the accuracy and reliability of detection.

[0090] At the same time, combined Figure 3-Figure 5 It can be seen (only the results of three different processes in this embodiment are used as examples), in the conventional double antibody sandwich mode ( Figure 3), after the antigen binds to the capture antibody, it can only connect to the detection antibody labeled with a fluorescent substance. Due to the limited number of fluorescein labeled on the detection antibody, the fluorescein labeling efficiency in the final complex is relatively low. For a single antigen, only three fluorescein labels can be achieved, that is, the labeling ratio of antigen to fluorescein is 1:3. In this mode, the amount of fluorescein labeling is relatively small. When detecting low-concentration NT-proBNP, the generated fluorescent signal is weak, resulting in limited detection sensitivity and difficulty in accurately detecting low-concentration samples. It is insufficient for the detection of low-concentration NT-proBNP in early disease diagnosis.

[0091] When using conventional streptavidin amplification mode ( Figure 4 ), the streptavidin membrane method is used. After the antigen is combined with the biotinylated capture antibody and the fluorescent substance labeled detection antibody, due to the limited number of fluorescein labeled on the detection antibody, the fluorescein labeling efficiency in the final complex is relatively low. For a single antigen, only 3 fluorescein labels can be achieved, that is, the labeling ratio of antigen to fluorescein is 1:3. However, compared with the conventional double antibody sandwich mode, this mode introduces streptavidin in the structure. In this mode, 4 antibodies can be connected to 2 antigens, and 4 antibodies are jointly connected to 1 streptavidin, and each streptavidin can be connected to 6 fluoresceins. This means that compared with the double antibody sandwich mode, the introduction of streptavidin increases the possibility of introducing fluorescein into the immune complex to a certain extent. Because streptavidin and biotin have a highly specific affinity, the biotinylated capture antibody and detection antibody can bind to streptavidin through biotin, making it possible to introduce the fluorescein linked to streptavidin into the complex, so that with the same amount of antigen, theoretically more fluorescein can participate in signal generation, so the generated fluorescent signal is slightly stronger than the conventional double antibody sandwich mode.

[0092] However, from the perspective of fluorescein labeling efficiency, due to factors such as the connection ratio of streptavidin to antibodies and steric hindrance, fluorescein labeling is relatively inefficient. On the one hand, the streptavidin membrane stripping operation is complicated, and it is difficult to accurately control the distribution and binding amount of streptavidin on the membrane, which requires high experimental conditions and is easily affected by fluctuations in experimental conditions, resulting in poor stability of the test results; on the other hand, although four antibodies are jointly linked to one streptavidin, in the actual binding process, steric hindrance may hinder streptavidin from fully exerting its ability to connect to fluorescein, so that not every streptavidin can fully achieve effective connection with six fluoresceins, and thus cannot fully utilize the amplification effect of streptavidin. When testing low-concentration samples, although it has a certain amplification effect compared to the double antibody sandwich mode, it still cannot meet the needs of high-sensitivity detection; and in the medium and high concentration range, its signal amplification effect is not ideal, and it cannot accurately reflect the changes in NT-proBNP concentration in the sample, affecting the accuracy and reliability of the test. For example, in medium and high concentration samples, since the fluorescence labeling efficiency is not optimal, as the antigen concentration increases, the growth rate of the fluorescence signal intensity is limited, and the signal amplification proportional to the antigen concentration cannot be well achieved, resulting in difficulty in accurate quantitative detection, which is not conducive to accurate assessment of the disease condition.

[0093] When the optimized biotin amplification mode of the present invention is adopted ( Figure 5 ), the capture antibody is used to draw the membrane, and the biotinylated detection antibody and the fluorescent substance labeled streptavidin are mixed in advance at the binding pad. In this mode, the fluorescein labeling efficiency is significantly improved in the final complex. For a single antigen, 6 fluorescein labels can be achieved, that is, the labeling ratio of antigen to fluorescein is 1:6, which greatly improves the fluorescein labeling efficiency. After the biotinylated detection antibody binds to the antigen, the streptavidin-fluorescein complex can quickly and efficiently bind to it through the highly specific affinity between biotin and streptavidin, thereby introducing more fluorescein into the immune complex. When detecting low-concentration NT-proBNP samples, it can generate a fluorescent signal that is significantly stronger than the previous two modes, effectively improving the detection sensitivity, and the minimum detectable is 20pg / L, meeting the clinical needs for low-concentration sample detection; in the medium and high concentration range, more fluorescein labels make the signal intensity increase more, and the detection signal is significantly improved, which can more accurately reflect the concentration changes of NT-proBNP in the sample, effectively expand the linear range of detection, and improve the accuracy and reliability of detection. At the same time, the mode of the present invention simplifies the operating process, avoids complicated operations such as streptavidin membrane stripping, reduces the requirements for the operating skills of experimenters, improves experimental efficiency and stability, and is more suitable for clinical rapid detection scenarios.

[0094] In summary, the optimized biotin amplification mode of the present invention shows excellent performance in NT-proBNP detection. It not only has high sensitivity in the low concentration range and can detect extremely low levels of NT-proBNP, which provides the possibility for early disease diagnosis; it can also provide accurate and reliable test results in the medium and high concentration range, with a good linear relationship, which is helpful for the assessment of the disease condition. In addition, compared with the conventional streptavidin amplification mode, the present invention reduces complex operations such as streptavidin membrane streaking in terms of operating steps, simplifies the experimental process, improves experimental efficiency, and is more conducive to application in clinical rapid detection scenarios.

[0095] Therefore, the biotin amplification mode of the present invention has significant advantages in NT-proBNP detection, and can effectively solve the problems existing in the prior art in terms of detection sensitivity, linear range and ease of operation, providing a more efficient and accurate detection method for clinical diagnosis, which has important application value and promotion prospects.

[0096] Example 3 Effects of different biotin types on the detection of NT-proBNP by the biotin amplification system of the present invention

[0097] The present invention is based on the principle of biotin-streptavidin synergistic amplification, and utilizes the highly specific affinity and multi-stage amplification effect between biotin and streptavidin to achieve high-sensitivity detection in the process of detecting NT-proBNP. Biotin is a key component, and its binding effect with the NT-proBNP labeled antibody directly affects the performance of the entire amplification system, and further affects the accuracy and sensitivity of the detection results. Therefore, in this embodiment, in order to further optimize the detection performance of the biotin amplification system, different types of biotin connected to the NT-proBNP labeled antibody were compared and screened, and the specific steps are as follows:

[0098] 1. Biotinylation of NT-proBNP labeled antibody

[0099] 1. Take 0.2 mg of NT-proBNP labeled antibody and add NHS-PEG12-Biotin at a molar ratio of antibody / activated biotin of 1:20, with a total volume of 100 μL;

[0100] 2. Incubate at room temperature for 30 minutes;

[0101] 3. Dialyze at 4°C for 72 hours and remove for later use.

[0102] 2. Biotin Type Information

[0103] Table 2. Information on different biotins

[0104] Biotin Thermo Fisher Scientific Part Number 1 EZ-Link Sulfo-NHS-LC-Biotin A39257 2 EZ-Link Sulfo-NHS-LC-LC-Biotin A35358 3 EZ-Link NHS-PEG12-Biotin A35389

[0105] By preparing NT-proBNP labeled antibodies labeled with different biotins and applying them to the biotin amplification system of the present invention for detection, the amplification effect data of different biotins at two concentrations of 20 pg / L and 5000 pg / L were obtained. The specific results are shown in Table 3:

[0106] Table 3. Amplification effects of different biotin

[0107]

[0108] 1. Analysis of low concentration samples (20pg / L)

[0109] In the detection of low-concentration NT-proBNP samples, there are obvious differences in the performance of different biotins. From the T / C value (representing the ratio of the signal intensity of the test line to the signal intensity of the quality control line, the higher the T / C value, the higher the detection sensitivity), biotin 3 (EZ-Link NHS-PEG12-Biotin) showed the highest T / C mean (0.098), which was significantly improved compared with the control group (0.039), and its coefficient of variation (CV) was 10.34%, indicating that this biotin has good stability and repeatability in low-concentration detection. The T / C mean of biotin 2 (EZ-Link Sulfo-NHS-LC-LC-Biotin) is 0.082, which is also higher than the control group, indicating that it has a certain amplification effect at low concentrations, but it is slightly inferior to biotin 3. The T / C mean of biotin 1 (EZ-Link Sulfo-NHS-LC-Biotin) is 0.042, which is higher than the control group, but the increase is not as obvious as biotin 3.

[0110] These results indicate that the structure and properties of biotin have an important influence on its binding to antibodies and subsequent amplification effects when detecting low-concentration NT-proBNP. Biotin 3 (EZ-Link NHS-PEG12-Biotin) has a small steric hindrance between biotin and antibodies due to the addition of PEG12 to increase the arm length between NHS and biotin, which allows the binding of more single-site streptavidin. The modified streptavidin can bind more fluorescein, thereby improving the detection sensitivity.

[0111] 2. Analysis of high concentration samples (5000pg / L)

[0112] In the detection of high-concentration samples, the difference in amplification effect of different biotins can also be observed. The mean T / C of biotin 3 reached 0.980, which was significantly higher than other biotins and the control group, and its CV value (6.17%) was relatively low, indicating that it not only had high signal intensity at high concentrations, but also had good accuracy and stability of the test results. The mean T / C of biotin 2 was 0.711, showing a certain amplification ability, but lower than biotin 3. The mean T / C of biotin 1 was 0.395, and the amplification effect at high concentrations was relatively weak.

[0113] This further proves that the choice of biotin type is crucial to the performance of the entire detection system in different concentration ranges. Biotin 3 (EZ-Link NHS-PEG12-Biotin) can also maintain good performance in high-concentration sample detection, which may be due to the fact that the complex formed after it binds to the antibody can more effectively promote the generation and amplification of fluorescent signals when interacting with streptavidin and antigen, thereby achieving more accurate quantitative detection.

[0114] Therefore, it can be seen that different types of biotin have a significant impact on the effect of the biotin amplification system of the present invention on the detection of NT-proBNP. Biotin 3 (EZ-Link NHS-PEG12-Biotin) showed the best comprehensive performance among the biotins tested in this experiment, and it has obvious advantages in terms of improving the detection sensitivity at low concentrations and ensuring the accuracy and stability of detection at high concentrations. Therefore, in the biotin amplification system of the present invention, the selection of biotin 3 (EZ-Link NHS-PEG12-Biotin) as the biotinylation reagent can significantly optimize the detection effect, provide strong support for the realization of high-sensitivity and high-accuracy NT-proBNP detection, and help improve the reliability and effectiveness of clinical diagnosis. At the same time, this study also provides valuable data and references for further understanding of the application of the relationship between the structure and function of biotin in immunoassays.

[0115] Example 4 Effect of different streptavidin types on the detection of NT-proBNP by the biotin amplification system of the present invention

[0116] The present invention is based on the principle of biotin-streptavidin synergistic amplification, and utilizes the multi-stage amplification effect generated by the specific binding between biotin and streptavidin to achieve high-sensitivity detection of NT-proBNP. Among them, streptavidin is a key component, and its complex with fluorescein after labeling plays an important role in signal conduction and amplification in the entire amplification system, which directly affects the accuracy and sensitivity of the detection results. Therefore, in this embodiment, in order to further optimize the detection performance of the biotin amplification system, different types of streptavidin connected to fluorescein were compared and screened, and the specific steps are as follows:

[0117] 1. The process of labeling fluorescein with streptavidin

[0118] 1. Add streptavidin and fluorescein at a ratio of 1 mg antibody / fluorescein: 10 μL, with a total volume of 400 μL;

[0119] 2. Incubate with rotation at room temperature for 3 hours, and add 10% BSA to block for 1 hour;

[0120] 3. After labeling, use the molecular sieve principle of chromatography column to remove excess fluorescein and collect the labeled complex.

[0121] 2. Streptavidin type information

[0122] Table 4. Information of different streptavidin

[0123] Streptavidin factory Part Number 1 Beijing Solable S9171 2 Thermo Fisher 434301 3 Weifang Bainodi BND56 4 Qingdao Henderson HDS-D1013 5 Jiangsu Boya A-SA-01 6 Hangzhou Bowo Biotech 22-003-01

[0124] By preparing different streptavidin-labeled fluorescein complexes and applying them to the biotin amplification system of the present invention for detection, the amplification effect data of different streptavidin at two concentrations of 20 pg / L and 5000 pg / L were obtained. The specific results are shown in Tables 5 and 6:

[0125] Table 5. Amplification effect of different streptavidin (1, 2, 3)

[0126]

[0127]

[0128] Table 6. Amplification effect of different streptavidin (4, 5, 6)

[0129]

[0130] 1. Analysis of low concentration samples (20pg / L)

[0131] In the detection of low-concentration NT-proBNP samples, the performance of different streptavidins showed obvious differences. From the perspective of T / C value, streptavidin 5 performed the best, with a T / C mean of 0.107, significantly higher than the control group (0.035), and a coefficient of variation (CV) of 6.08%, indicating that this streptavidin has excellent stability and repeatability in low-concentration detection, can effectively enhance the detection signal, and is more sensitive to the detection of low-concentration NT-proBNP. The T / C mean values ​​of streptavidin 1 and streptavidin 2 were 0.042, respectively. Although they were also higher than the control group, the amplification effect was significantly lower than that of streptavidin 5, and the CV value of streptavidin 1 (8.28%) was relatively high, and its stability was slightly inferior. The mean T / C of streptavidin 3 (0.040, the mean T / C of streptavidin 6 is 0.039, which is similar to streptavidin 1 and 2, but lower than streptavidin 5, and the advantage in low-concentration detection is not obvious. The mean T / C of streptavidin 4 is 0.038, which is relatively weak, and its CV value (12.05%) is high, and the stability is poor, which may cause large fluctuations in the results when detecting at low concentrations.

[0132] These results fully demonstrate that in the low-concentration NT-proBNP detection scenario, the source and properties of streptavidin have a crucial impact on its ability to bind to biotinylated antibodies and fluorescein, as well as the subsequent signal amplification effect. Streptavidin produced by different manufacturers may differ in purity, activity, compatibility with other reagents, and spatial structure, which in turn leads to different performance in the detection system.

[0133] Among them, single-site or double-site streptavidin can bind more fluorescein after modification. In the present invention, streptavidin 5 is a single binding site. Compared with conventional streptavidin with four binding sites, when binding with biotinylated antibodies and fluorescein, there is no competition caused by too many sites, which leads to reduced sensitivity. In the low-concentration NT-proBNP detection scenario, this structural characteristic enables streptavidin 5 to bind to biotin on the biotinylated antibody more accurately, avoiding the problem of reduced binding efficiency caused by site competition, thereby effectively enhancing the binding ability with the biotinylated antibody, laying a good foundation for subsequent signal amplification.

[0134] 2. Analysis of high concentration samples (5000pg / L)

[0135] In the detection of high-concentration samples, streptavidin 5 still showed excellent performance, with a T / C mean of 0.984, much higher than the control group (0.356), and a CV value of only 3.91%, which means that at high concentrations, not only can it produce extremely strong detection signals, but the detection results are also highly accurate and stable, which can provide a reliable basis for clinical diagnosis. The T / C mean values ​​of streptavidin 3 and streptavidin 6 were 0.409 and 0.376, respectively, which were higher than the control group, showing a certain amplification effect, but there was still a large gap compared with streptavidin 5. The T / C mean values ​​of streptavidin 1, streptavidin 2, and streptavidin 4 were 0.377, 0.373, and 0.379, respectively, which were relatively close, and the CV values ​​were 5.50%, 6.58%, and 6.38%, respectively. They had relatively stable performance in high-concentration detection and could meet certain detection needs, but the amplification effect was relatively weak.

[0136] This further emphasizes the key impact of the choice of streptavidin type on the performance of the entire detection system at different concentration ranges. Suitable streptavidin can synergize with other reagents in high-concentration samples to more efficiently promote signal amplification, thereby improving the accuracy and reliability of detection. This may be closely related to its own structural characteristics, active state after binding to fluorescein, interaction efficiency with biotinylated antibodies, and stability in the entire detection system.

[0137] Therefore, it can be seen that different types of streptavidin have significant and diverse effects on the effect of the biotin amplification system of the present invention in detecting NT-proBNP. Among the streptavidin screened in this embodiment, the single-binding site streptavidin 5 showed extremely outstanding comprehensive performance in both low-concentration and high-concentration sample detection, and was significantly better than other streptavidin in terms of T / C value and stability. Streptavidin 5 can bind more fluorescein after modification. During the detection process, when streptavidin 5 binds to the biotin on the biotinylated labeled antibody, more fluorescein it carries can be introduced into the immune complex, significantly enhancing the intensity of the fluorescent signal. When detecting low-concentration samples, a stronger fluorescent signal makes the detection more sensitive and can effectively improve the detection sensitivity; in high-concentration sample detection, it also helps to generate a stronger and more stable detection signal, ensuring the accuracy and stability of the detection results, thereby showing excellent performance over the entire concentration range.

[0138] Therefore, in the biotin amplification system of the present invention, the single binding site structural characteristics of streptavidin 5 and its high binding ability to fluorescein enable it to perform well in the binding process with biotinylated antibodies and fluorescein, effectively improving the signal amplification effect, and then showing extremely outstanding comprehensive performance in both low-concentration and high-concentration NT-proBNP detection, becoming the preferred streptavidin in the biotin amplification system of the present invention. The preferred streptavidin 5 can significantly optimize the detection effect, effectively realize high-sensitivity and high-accuracy NT-proBNP detection, provide solid and reliable technical support for clinical diagnosis, and also provide a scientific and accurate reference basis for the subsequent application of streptavidin in the detection system, which is helpful to promote the further development and improvement of detection technology in this field.

[0139] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A detection kit based on biotin-streptavidin amplification mode, characterized in that: The invention comprises a test strip, which comprises a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad. The nitrocellulose membrane is provided with a detection line and a quality control line. The conjugation pad comprises a labeled antibody, streptavidin, fluorescein and biotin. The detection line comprises a coated antibody, and the quality control line is coated with goat anti-rabbit IgG.

2. The detection kit according to claim 1, characterized in that The labeled antibody is labeled with biotin to form a biotinylated labeled antibody, and the streptavidin is coupled with fluorescein to form streptavidin-fluorescein.

3. The detection kit according to claim 2, characterized in that The biotinylated labeled antibody specifically recognizes the antigen that has been bound to the coated antibody to form an immune complex of "coated antibody-antigen-biotinylated labeled antibody".

4. The detection kit according to claim 3, characterized in that The streptavidin-fluorescein is combined with the biotin on the biotinylated labeled antibody, and the fluorescein is introduced into the immune complex to achieve multiple amplification of the fluorescence signal.

5. The detection kit according to claim 4, characterized in that The coated antibody includes NT-proBNP coated antibody, and the labeled antibody includes NT-proBNP labeled antibody.

6. The detection kit according to claim 5, characterized in that The molar ratio of the labeled antibody to biotin is 1:10-1:

30.

7. The detection kit according to claim 5, characterized in that The molar ratio of fluorescent streptomycin to biotin is 4:1-1:

1.

8. A method for detecting NT-proBNP, characterized in that: NT-proBNP is detected using the kit according to any one of claims 1 to 7.

9. The detection method according to claim 8, characterized in that: The following steps are involved: (1) Add the sample to be tested to the sample pad and allow the sample to migrate on the test strip; (2) The NT-proBNP antigen in the sample binds to the NT-proBNP monoclonal antibody on the nitrocellulose membrane and reacts with the biotinylated NT-proBNP labeled antibody and streptavidin-conjugated fluorescein complex on the binding pad to form an immune complex; (3) Detect the fluorescence signal intensity on the immune complex and determine the NT-proBNP content in the sample based on the standard curve.

10. Use of an immune complex for preparing a reagent for improving the sensitivity and accuracy of antigen detection, characterized in that: The immune complex includes an immune complex of "coated antibody-antigen-biotinylated labeled antibody". Through the binding of streptavidin-fluorescein to biotin on the biotinylated labeled antibody, fluorescein is introduced into the immune complex to achieve multiple amplification of fluorescent signals.

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