Substrate solution suitable for full-automatic immunoassay system and preparation method thereof

Abstract

The invention discloses a substrate solution suitable for a full-automatic immunoassay system and a preparation method of the substrate solution, and relates to the technical field of in-vitro diagnostic reagents. The substrate solution is composed of a solution A and a solution B, wherein the solution A contains a luminol derivative (1.0-3.0 g / L), a buffering agent (10-30 g / L) and an antibacterial agent (0.05-0.5 g / L), and the pH value of the solution A is 9.00; the solution B contains urea peroxide (0.5 g / L to 2.0 g / L), N-(3-chloro-4-hydroxyphenyl) acetamide (0.1 g / L to 0.5 g / L), a buffering agent (0.5 g / L to 3.0 g / L), an antibacterial agent (0.05 g / L to 0.5 g / L) and a peroxide stabilizer (0.5 g / L to 5.0 g / L), and the pH value of the solution B is 5.20. A luminol and urea peroxide luminescent system is adopted, and N-(3-chloro-4-hydroxyphenyl) acetamide is used as a reinforcing agent, so that the luminescent intensity and stability are improved, and the validity period of a substrate solution is prolonged to 12 months. The preparation method is simple, low in cost, suitable for a full-automatic immunoassay system, capable of effectively improving the detection sensitivity and stability, capable of replacing imported reagents and capable of reducing the clinical examination cost.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, in particular to a substrate solution suitable for a full-automatic immunoassay system and a preparation method thereof. Background Art

[0002] Chemiluminescence immunoassay (CLIA) is the fastest-growing and most advanced labeled immunoassay technology in recent years. Due to its unique performance advantages, it has become one of the core technologies in the field of clinical testing. Compared with traditional biological detection technologies, chemiluminescence immunoassay has the following significant advantages: High sensitivity: It can detect extremely low concentrations of target substances and is suitable for clinical projects that require high-precision detection. Strong specificity: Through the specific binding of antigens and antibodies, the high accuracy of the test results is ensured. Stable and rapid method: The reaction process is stable and the detection time is short, meeting the needs of rapid clinical diagnosis. Wide detection range: It is suitable for the detection of multiple biomarkers, covering a wide range from hormones to tumor markers. Multi-item detection: It supports the simultaneous detection of multiple indicators to improve efficiency.

[0003] Based on these advantages, chemiluminescence immunoassay analyzers have become the most widely used and most actively developed equipment in clinical testing, and are gradually replacing traditional biological detection technologies such as enzyme-linked immunosorbent assay (ELISA). They are widely used in infectious disease screening, endocrine testing, myocardial marker analysis and other fields.

[0004] The core of chemiluminescent immunoassay technology lies in its luminescence detection principle, which primarily includes the following three systems: The electrochemiluminescence system, which generates a light signal by exciting a luminescent substance with an electrode, offers a high signal-to-noise ratio and controllability, and is commonly used in high-end analytical instruments. Luminol (Luminol) was the first luminescent substance used in chemiluminescent immunoassays. In this system, antibodies are typically labeled with a peroxidase (such as horseradish peroxidase, HRP). After the immunoreaction is complete, luminol acts as a luminescent substrate, undergoing oxidation under the catalysis of peroxidase and alkaline hydrogen peroxide, releasing photons. The luminescence intensity is proportional to the enzyme concentration, indirectly reflecting the target analyte content. Acridinium esters emit light directly under the action of hydrogen peroxide, eliminating the need for enzyme catalysis. This rapid and simple method makes them suitable for certain specific applications. Luminol systems have been widely studied and applied due to their low cost and mild reaction conditions. However, early luminol luminescence systems suffered from significant drawbacks: The flash of light lasts only a few seconds, making the signal transient and difficult to capture. Low luminous intensity: The light signal is weak and cannot meet the requirements of high-sensitivity detection. Difficulty in measurement: The instantaneous nature and low intensity make instrument measurement complex, affecting the reliability of the results.

[0005] To overcome the limitations of earlier systems, researchers have introduced luminescence enhancers, such as p-iodophenol (PIP), into the luminol luminescence system. These enhancers improve reaction efficiency, enhance the luminescence signal, and prolong signal stability, thereby improving detection sensitivity and accuracy. However, despite the wide variety of luminescence enhancers, most still suffer from the following issues: High background signal: Enhancers may increase nonspecific luminescence, reducing the signal-to-noise ratio. Poor luminescence stability: Although the signal is enhanced, it is difficult to maintain stability over long periods of detection. Difficulty in balancing intensity and stability: Existing enhancers often only optimize one aspect of performance and cannot simultaneously meet the requirements of high intensity and long-term stability. These shortcomings directly affect the sensitivity and precision of detection, limiting the application of luminol luminescence systems in demanding clinical testing. Furthermore, the poor stability of aqueous hydrogen peroxide is a major bottleneck. As a key reagent in the luminescence reaction, hydrogen peroxide easily decomposes during storage and has a short shelf life (typically only a few months), further hindering the widespread adoption of the luminol system.

[0006] Among the numerous chemiluminescence immunoassay analyzer brands, the Ortho VITROS 3600 fully automated immunoassay system holds a significant market position thanks to its advanced technology. Its key features include: Dry chemistry technology: Requires no liquid handling, reducing sample contamination and external interference. Proprietary enhanced chemiluminescence technology: Optimizes the luminescence system, providing higher signal intensity and stability. Ease of operation: The fully automated design reduces manual intervention and improves testing efficiency. Reliable results: It excels in detecting infectious diseases (such as hepatitis B and HIV) and cardiac markers (such as troponin). These advantages have earned it a high market share in China, making it particularly popular in clinical scenarios requiring high precision and rapid results. However, the system also has significant shortcomings: The substrate solution and reagents rely on imports: All original imported reagents are used, resulting in high procurement costs. A short shelf life: The reagents are labeled for a six-month shelf life, but due to the logistics cycle from foreign production to domestic sales, the actual shelf life is often shortened to three months. Inconvenience: Ordering in large quantities can easily lead to waste due to expiration. Ordering in small quantities can delay testing due to insufficient supply, impacting clinical practice. These problems not only increase the financial burden on medical institutions, but also reduce the flexibility and efficiency of the testing process. Summary of the Invention

[0007] To address the challenges in the aforementioned technologies and applications, the present invention proposes a novel substrate solution suitable for fully automated immunoassay systems and a method for its preparation, aiming to improve existing systems in the following aspects: Improving luminescence intensity: By optimizing the luminescence system formula, the light signal output of luminol or other luminescent substances is enhanced. Improving stability: Improving enhancers or adding stabilizers prolongs the duration of the luminescence signal and reduces the background signal. Extending shelf life: Optimizing the storage stability of key components such as hydrogen peroxide extends the shelf life of the substrate solution to more than 12 months. Reducing production costs: Utilizing domestically produced raw materials and technologies reduces dependence on imported reagents, thereby lowering procurement and usage costs. This solution not only enhances the detection sensitivity and precision of chemiluminescent immunoassay technology but also provides a more efficient and economical tool for clinical testing. For example, the improved substrate solution is seamlessly compatible with fully automated systems such as the Ortho VITROS 3600, addressing the issues of short shelf life and high cost of reagents, and providing users with more flexible inventory management and more reliable testing support. Chemiluminescent immunoassay technology has become a mainstream technology for clinical testing due to its high sensitivity, strong specificity, rapidity, and stability. Although the luminol luminescence system was limited by its early defects and reagent stability issues, these shortcomings are gradually being overcome through the development of luminescence enhancers and novel substrate solutions. The application of advanced equipment such as the Ortho VITROS 3600 has further promoted the popularization of this technology, and the improved solutions proposed in this invention have injected new vitality into this field. In the future, with the continuous optimization of the luminescence system and the advancement of localization, chemiluminescence immunoassay technology will play a greater role in clinical diagnosis, providing patients with more accurate and convenient testing services.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A substrate solution suitable for a fully automatic immunoassay system comprises a solution A and a solution B. The solution A comprises: 1.0 g / L to 3.0 g / L of a luminol derivative, 10 g / L to 30 g / L of a buffer, 0.05 g / L to 0.5 g / L of an antibacterial agent, and a pH value of 9.00; and the solution B comprises: 0.5 g / L to 2.0 g / L of urea peroxide, 0.1 g / L to 0.5 g / L of N-(3-chloro-4-hydroxyphenyl)acetamide, 0.5 g / L to 3.0 g / L of a buffer, 0.05 g / L to 0.5 g / L of an antibacterial agent, 0.5 g / L to 5.0 g / L of a peroxide stabilizer, and a pH value of 5.20.

[0010] Preferably, in solution A, the luminol derivative is one of luminol, luminol monosodium salt, and isoluminol.

[0011] Preferably, in solution A, the buffer is one or more of Tris buffer, borax, triethanolamine, imidazole, and 3-amino-1-propanol.

[0012] Preferably, in liquid A, the antibacterial agent is selected from one or more of ProClin 150, ProClin 200, ProClin 300, and ProClin 950.

[0013] Preferably, in solution B, the buffer is one or more of formic acid, acetic acid, succinic acid, and citric acid.

[0014] Preferably, in liquid B, the peroxide stabilizer is one or more of boric acid, borax, 8-hydroxyquinoline, urea, sodium polyacrylate, and disodium ethylenediaminetetraacetic acid.

[0015] As a further technical solution, the substrate solution includes the following components:

[0016] The substrate solution A comprises 2.35 g / L of luminol derivative, 26.7 g / L of Tris buffer, 0.5 g / L of 3-amino-1-propanol, and 0.25 g / L of ProClin 3000, with a pH of 9.00.

[0017] The components of the substrate solution B are 1.54 g / L of urea peroxide, 0.38 g / L of N-(3-chloro-4-hydroxyphenyl)acetamide, 1.5 g / L of acetic acid, 0.25 g / L of ProClin 3000, 1.5 g / L of boric acid, 0.15 g / L of 8-hydroxyquinoline, and 3.2 g / L of urea, with a pH value of 5.20.

[0018] The method for preparing a substrate solution suitable for a fully automatic immunoassay system as described above comprises the following steps:

[0019] (1) Preparation of Solution A: Weighing the raw materials: Accurately weigh the luminol derivative, buffer, and antimicrobial agent according to the concentration range described in claim 1; Dissolving: Add the weighed luminol derivative, buffer, and antimicrobial agent to deionized water and stir thoroughly until completely dissolved; Adjusting the pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 9.00; Filtering: Filter the solution to remove any insoluble matter to obtain a clear Solution A; Aliquoting and storing: Aliquot the prepared Solution A into clean, sealed containers to avoid contamination;

[0020] (2) Preparation of Solution B: Weighing raw materials: Accurately weigh urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer according to the concentration range of claim 1; Dissolving: Add the weighed urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer into deionized water and stir thoroughly until completely dissolved; Adjusting pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 5.20; Filtering: Filter the solution to remove insoluble matter to obtain a clear Solution B; Aliquoting and storing: Aliquot the prepared Solution B into clean, sealed containers to avoid contamination;

[0021] (4) Storage and use: Store solution A and solution B separately in a dark environment at 2-8°C. When using, according to the operating procedures of the fully automatic immunoassay system, mix solution A and solution B as required or add them separately to the system for testing.

[0022] Beneficial effects

[0023] The present invention comprehensively optimizes the formula of the luminol luminescent substrate solution and significantly improves the stability of the product by selecting carbamide peroxide as the oxidant. Compared with hydrogen peroxide commonly used in existing imported reagents, carbamide peroxide has better chemical stability and is not easy to decompose at room temperature, avoiding the performance degradation caused by the decomposition of hydrogen peroxide during storage. This improvement extends the shelf life of the substrate solution from 6 months of imported reagents to 12 months, which not only enhances the practicality of the product but also reduces the economic burden caused by frequent reagent replacement. In terms of luminescence performance, the present invention introduces N-(3-chloro-4-hydroxyphenyl)acetamide as a luminescence enhancer. Compared with the phenol enhancers used in traditional technologies, its luminescence intensity is increased by nearly 2 times and is also better than the common aniline enhancers. This new enhancer significantly improves the luminescence efficiency by optimizing the luminescence reaction pathway of luminol and extends the duration of the luminescence signal, ensuring that the luminescence value remains stable during the detection process. This technological breakthrough significantly improves the sensitivity and precision of the detection, enabling the substrate solution of the present invention to completely replace imported reagents in terms of performance. In addition, the present invention also achieves important improvements in environmental protection. By adopting environmentally friendly antimicrobial agents (such as the ProClin series) to replace sodium azide commonly used in imported reagents, environmental and health risks are effectively reduced. Sodium azide, as a toxic substance, seriously pollutes the environment and poses a safety hazard, while the ProClin series of antimicrobial agents have the characteristics of low toxicity and environmental protection, meet the development requirements of green chemistry, and ensure the safety and sustainability of the product. At the production and application level, the present invention simplifies the production process and significantly reduces manufacturing costs by optimizing the formula and preparation process. This not only makes the substrate solution more competitive in price, but also significantly reduces the overall cost of clinical testing and alleviates the burden on medical institutions. At the same time, the localized production of the present invention reduces dependence on imported reagents, enhances the independent innovation capability of my country's in vitro diagnostic reagent industry, and is of great significance to promoting the sustainable development of the biopharmaceutical industry.

[0024] In summary, the innovative design of the oxidant, luminescence enhancer, and antimicrobial agent significantly improves the stability, luminescence performance, and environmental friendliness of the luminol luminescent substrate solution, while simultaneously reducing production and usage costs. This technological breakthrough and practical advantages not only enable it to fully replace imported reagents but also demonstrate broad market application prospects and industry-driven value. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Example 1

[0027] The substrate solutions for the Ortho VITROS 3600 automated immunoassay system include Substrate Solution A and Substrate Solution B, each containing the following components: Substrate Solution A consists of a luminol derivative, a buffer, and a preservative (ProClin 300); Substrate Solution B consists of urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, a buffer, a preservative (ProClin 300), and a peroxide stabilizer. The pH is 5.20. In Substrate Solution A, the luminol derivative is luminol, the buffer is Tris buffer (Tris-HCl) and 3-amino-1-propanol, and the antibacterial agent is ProClin 300. Substrate Solution B uses acetic acid as the buffer and ProClin 300 as the antibacterial agent. Peroxide stabilizers include boric acid, 8-hydroxyquinoline, and urea. The contents of the above components are as follows: Substrate Solution A comprises 1.85 g / L of a luminol derivative, 18.5 g / L of Tris buffer, 9.0 g / L of 3-amino-1-propanol, and 0.25 g / L of ProClin 3000, with a pH of 9.00; Substrate Solution B comprises 0.95 g / L of urea peroxide, 0.25 g / L of N-(3-chloro-4-hydroxyphenyl)acetamide, 1.0 g / L of acetic acid, 0.25 g / L of ProClin 3000, 2.3 g / L of boric acid, 0.18 g / L of 8-hydroxyquinoline, and 1.6 g / L of urea, with a pH of 5.20. Substrate Solution A prepared in Example 1 has a pH of 9.00 at 25°C ± 0.1°C, a conductivity of 5.78 ms / cm, and an absorbance of 0.278 at 301 nm when diluted 10-fold by volume. The pH value of substrate solution B is 5.20 at 25℃±0.1℃, and the conductivity is 12.80ms / cm.

[0028] The method for preparing the substrate solution for the Ortho VITROS 3600 fully automatic immunoassay system comprises the following steps:

[0029] (1) Preparation of Solution A: Weighing the raw materials: Accurately weigh the luminol derivative, buffer, and antimicrobial agent according to the concentration range described in claim 1; Dissolving: Add the weighed luminol derivative, buffer, and antimicrobial agent to deionized water and stir thoroughly until completely dissolved; Adjusting the pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 9.00; Filtering: Filter the solution to remove any insoluble matter to obtain a clear Solution A; Aliquoting and storing: Aliquot the prepared Solution A into clean, sealed containers to avoid contamination;

[0030] (2) Preparation of Solution B: Weighing raw materials: Accurately weigh urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer according to the concentration range of claim 1; Dissolving: Add the weighed urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer into deionized water and stir thoroughly until completely dissolved; Adjusting pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 5.20; Filtering: Filter the solution to remove insoluble matter to obtain a clear Solution B; Aliquoting and storing: Aliquot the prepared Solution B into clean, sealed containers to avoid contamination;

[0031] (4) Storage and use: Store solution A and solution B separately in a dark environment at 2-8°C. When using, according to the operating procedures of the fully automatic immunoassay system, mix solution A and solution B as required or add them separately to the system for testing.

[0032] Example 2

[0033] The substrate solutions for the Ortho VITROS 3600 fully automated immunoassay system include Substrate Solution A and Substrate Solution B, each containing the following components: Substrate Solution A consists of a luminol derivative, a buffer, and a preservative; Substrate Solution B consists of urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, a buffer, a preservative, and a peroxide stabilizer. The pH is 5.20. The luminol derivative in Substrate Solution A is luminol, the buffer is Tris buffer and 3-amino-1-propanol, and the antibacterial agent is ProClin 300. Substrate Solution B uses acetic acid as the buffer and ProClin 300 as the antibacterial agent. Peroxide stabilizers include boric acid, 8-hydroxyquinoline, and urea. The contents of the above components are as follows: Substrate Solution A comprises 1.85 g / L of a luminol derivative, 18.5 g / L of Tris buffer, 9.0 g / L of 3-amino-1-propanol, and 0.25 g / L of ProClin 3000, with a pH of 9.00; Substrate Solution B comprises 0.95 g / L of urea peroxide, 0.25 g / L of N-(3-chloro-4-hydroxyphenyl)acetamide, 1.0 g / L of acetic acid, 0.25 g / L of ProClin 3000, 2.3 g / L of boric acid, 0.18 g / L of 8-hydroxyquinoline, and 1.6 g / L of urea, with a pH of 5.20. Substrate Solution A prepared in Example 2 has a pH of 9.05 at 25°C ± 0.1°C, a conductivity of 5.87 ms / cm, and an absorbance of 0.283 at 301 nm when diluted 10-fold by volume. The pH value of substrate solution B is 5.24 at 25℃±0.1℃, and the conductivity is 13.56ms / cm.

[0034] The method for preparing the substrate solution for the Ortho VITROS 3600 fully automatic immunoassay system comprises the following steps:

[0035] (1) Preparation of Solution A: Weighing the raw materials: Accurately weigh the luminol derivative, buffer, and antimicrobial agent according to the concentration range described in claim 1; Dissolving: Add the weighed luminol derivative, buffer, and antimicrobial agent to deionized water and stir thoroughly until completely dissolved; Adjusting the pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 9.00; Filtering: Filter the solution to remove any insoluble matter to obtain a clear Solution A; Aliquoting and storing: Aliquot the prepared Solution A into clean, sealed containers to avoid contamination;

[0036] (2) Preparation of Solution B: Weighing raw materials: Accurately weigh urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer according to the concentration range of claim 1; Dissolving: Add the weighed urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer into deionized water and stir thoroughly until completely dissolved; Adjusting pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 5.20; Filtering: Filter the solution to remove insoluble matter to obtain a clear Solution B; Aliquoting and storing: Aliquot the prepared Solution B into clean, sealed containers to avoid contamination;

[0037] (4) Storage and use: Store solution A and solution B separately in a dark environment at 2-8°C. When using, according to the operating procedures of the fully automatic immunoassay system, mix solution A and solution B as required or add them separately to the system for testing.

[0038] Example 3

[0039] The substrate solutions for the Ortho VITROS 3600 fully automated immunoassay system include Substrate Solution A and Substrate Solution B, each containing the following components: Substrate Solution A consists of a luminol derivative, a buffer, and a preservative; Substrate Solution B consists of urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, a buffer, a preservative, and a peroxide stabilizer. The pH is 5.20. The luminol derivative in Substrate Solution A is luminol, the buffer is Tris buffer and 3-amino-1-propanol, and the antibacterial agent is ProClin 300. Substrate Solution B uses acetic acid as the buffer and ProClin 300 as the antibacterial agent. Peroxide stabilizers include boric acid, 8-hydroxyquinoline, and urea. The contents of the above components are as follows: Substrate Solution A comprises 2.35 g / L of a luminol derivative, 22.5 g / L of a Tris buffer, 9.0 g / L of 3-amino-1-propanol, and 0.25 g / L of ProClin 3000, with a pH of 9.00; Substrate Solution B comprises 1.54 g / L of urea peroxide, 0.38 g / L of N-(3-chloro-4-hydroxyphenyl)acetamide, 1.5 g / L of acetic acid, 0.25 g / L of ProClin 3000, 1.5 g / L of boric acid, 0.15 g / L of 8-hydroxyquinoline, and 3.2 g / L of urea, with a pH of 5.20. Substrate Solution A prepared in Example 3 has a pH of 8.91 at 25°C ± 0.1°C, a conductivity of 5.68 ms / cm, and an absorbance of 0.277 at 301 nm when diluted 10-fold by volume. The pH value of substrate solution B is 5.13 at 25℃±0.1℃, and the conductivity is 12.76ms / cm.

[0040] The method for preparing the substrate solution for the Ortho VITROS 3600 fully automatic immunoassay system comprises the following steps:

[0041] (1) Preparation of Solution A: Weighing the raw materials: Accurately weigh the luminol derivative, buffer, and antimicrobial agent according to the concentration range described in claim 1; Dissolving: Add the weighed luminol derivative, buffer, and antimicrobial agent to deionized water and stir thoroughly until completely dissolved; Adjusting the pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 9.00; Filtering: Filter the solution to remove any insoluble matter to obtain a clear Solution A; Aliquoting and storing: Aliquot the prepared Solution A into clean, sealed containers to avoid contamination;

[0042] (2) Preparation of Solution B: Weighing raw materials: Accurately weigh urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer according to the concentration range of claim 1; Dissolving: Add the weighed urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer into deionized water and stir thoroughly until completely dissolved; Adjusting pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 5.20; Filtering: Filter the solution to remove insoluble matter to obtain a clear Solution B; Aliquoting and storing: Aliquot the prepared Solution B into clean, sealed containers to avoid contamination;

[0043] (4) Storage and use: Store solution A and solution B separately in a dark environment at 2-8°C. When using, according to the operating procedures of the fully automatic immunoassay system, mix solution A and solution B as required or add them separately to the system for testing.

[0044] Example 4

[0045] The substrate solutions for the Ortho VITROS 3600 fully automated immunoassay system include Substrate Solution A and Substrate Solution B, each containing the following components: Substrate Solution A consists of a luminol derivative, a buffer, and a preservative; Substrate Solution B consists of urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, a buffer, a preservative, and a peroxide stabilizer. The pH is 5.20. The luminol derivative in Substrate Solution A is luminol, the buffer is Tris buffer and 3-amino-1-propanol, and the antibacterial agent is ProClin 300. Substrate Solution B uses acetic acid as the buffer and ProClin 300 as the antibacterial agent. Peroxide stabilizers include boric acid, 8-hydroxyquinoline, and urea. The contents of the above components are as follows: Substrate Solution A comprises 1.85 g / L of a luminol derivative, 18.5 g / L of Tris buffer, 7.4 g / L of 3-amino-1-propanol, and 0.25 g / L of ProClin 3000, with a pH of 9.00; Substrate Solution B comprises 0.96 g / L of urea peroxide, 0.25 g / L of N-(3-chloro-4-hydroxyphenyl)acetamide, 1.5 g / L of acetic acid, 0.25 g / L of ProClin 3000, 1.5 g / L of boric acid, 0.15 g / L of 8-hydroxyquinoline, and 3.2 g / L of urea, with a pH of 5.20. Substrate Solution A prepared in Example 4 has a pH of 8.95 at 25°C ± 0.1°C, a conductivity of 5.68 ms / cm, and an absorbance of 0.279 at 301 nm when diluted 10-fold by volume. The pH value of substrate solution B is 5.20 at 25℃±0.1℃, and the conductivity is 12.81ms / cm.

[0046] The method for preparing the substrate solution for the Ortho VITROS 3600 fully automatic immunoassay system comprises the following steps:

[0047] (1) Preparation of Solution A: Weighing the raw materials: Accurately weigh the luminol derivative, buffer, and antimicrobial agent according to the concentration range described in claim 1; Dissolving: Add the weighed luminol derivative, buffer, and antimicrobial agent to deionized water and stir thoroughly until completely dissolved; Adjusting the pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 9.00; Filtering: Filter the solution to remove any insoluble matter to obtain a clear Solution A; Aliquoting and storing: Aliquot the prepared Solution A into clean, sealed containers to avoid contamination;

[0048] (2) Preparation of Solution B: Weighing raw materials: Accurately weigh urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer according to the concentration range of claim 1; Dissolving: Add the weighed urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent, and peroxide stabilizer into deionized water and stir thoroughly until completely dissolved; Adjusting pH: Monitor the pH value of the solution using a pH meter and adjust the pH to 5.20; Filtering: Filter the solution to remove insoluble matter to obtain a clear Solution B; Aliquoting and storing: Aliquot the prepared Solution B into clean, sealed containers to avoid contamination;

[0049] (4) Storage and use: Store solution A and solution B separately in a dark environment at 2-8°C. When using, according to the operating procedures of the fully automatic immunoassay system, mix solution A and solution B as required or add them separately to the system for testing.

[0050] Through specific ratios and preparation processes, the prepared substrate solution for the Ortho VITROS 3600 fully automatic immunoassay system improves product stability during use, extends its shelf life to 12 months, has a high luminescence value, a long plateau period, and better sensitivity and precision of test results. The reagents are more environmentally friendly and safer to use. The production process is simple and the cost is low, making it a complete substitute for imported reagents.

[0051] Performance test of the substrate solution used in the present invention and the Ortho VITROS 3600 fully automatic immunoassay system:

[0052] Sensitivity verification:

[0053] The substrate solutions A and B prepared in Examples 1-3 were placed in the corresponding positions of the Ortho VITROS 3600 fully automated immunoassay system for validation testing. Serum from 40 patients was tested, and the sensitivity and correlation of the results were compared with those obtained with the original reagents. The following table shows the statistical results:

[0054] Table 1: Comparison of the accuracy of Example 1 and the original supporting reagents MYOG (myoglobin), TNI (troponin), and NBNP2 (N-terminal brain natriuretic peptide)

[0055]

[0056]

[0057]

[0058] Table 2: Comparison of the accuracy of Example 1 and the original supporting reagents PROG (progesterone), SYPH (syphilis antibody), and HIV (human immunodeficiency virus type 1+2 combined antibody)

[0059]

[0060]

[0061]

[0062] Table 3: Correlation between the test results of Example 1 and the original reagent

[0063]

[0064] The results showed that the detection results of the self-prepared substrate solution A and substrate solution B in Example 1 had a good correlation with the accuracy of the original reagents, and the results were accurate and reliable.

[0065] Table 4: Comparison of the accuracy of Example 2 and the original supporting reagents MYOG (myoglobin), TNI (troponin), and NBNP2 (N-terminal brain natriuretic peptide)

[0066]

[0067]

[0068]

[0069] Table 5: Comparison of the accuracy of Example 2 and the original supporting reagents PROG (progesterone), SYPH (syphilis antibody), and HIV (human immunodeficiency virus type 1+2 combined antibody)

[0070]

[0071]

[0072]

[0073] Table 6: Correlation between the test results of Example 2 and the original reagent

[0074]

[0075]

[0076] The results showed that the detection results of the self-prepared substrate solution A and substrate solution B in Example 2 had a good correlation with the accuracy of the original reagents, and the results were accurate and reliable.

[0077] Table 7: Comparison of the accuracy of Example 3 and the original supporting reagents MYOG (myoglobin), TNI (troponin), and NBNP2 (N-terminal brain natriuretic peptide)

[0078]

[0079]

[0080]

[0081] Table 8: Comparison of the accuracy of Example 3 and the original supporting reagents PROG (progesterone), SYPH (syphilis antibody), and HIV (human immunodeficiency virus type 1+2 combined antibody)

[0082]

[0083]

[0084] Table 9: Correlation between the test results of Example 3 and the original reagent

[0085]

[0086] The results show that the test results of the self-prepared substrate liquid A and substrate liquid B in Example 3 have a good correlation with the accuracy of the original reagents, and the results are accurate and reliable. In summary, the substrate liquids for the Ortho VITROS 3600 fully automatic immunoassay system in Examples 1-3 have a good correlation with the accuracy of the test results of the original reagents, indicating that the self-prepared reagents can still complete the measurement on the Ortho VITROS 3600 fully automatic immunoassay system analyzer on the basis of changing the formula, and the results are accurate and reliable.

[0087] Precision verification:

[0088] Take one portion of normal serum and repeat the measurement 11 times using the self-prepared Example 1 and the original supporting reagent, respectively, and compare the precision of the measurement results with that of the original supporting reagent.

[0089] Table 10: Comparison of the precision of Example 1 and the original supporting reagents MYOG (myoglobin), TNI (troponin), NBNP2 (N-terminal brain natriuretic peptide), and PROG (progesterone)

[0090]

[0091]

[0092] Table 11: The precision comparison results of Example 1 and the original reagents SYPH (Syphilis Treponema pallidum antibody) and HIV (Human Immunodeficiency Virus Type 1+2 combined antibody) are as follows:

[0093]

[0094]

[0095] Table 12: Comparison of the precision of Example 2 and the original supporting reagents MYOG (myoglobin), TNI (troponin), NBNP2 (N-terminal brain natriuretic peptide), and PROG (progesterone)

[0096] Table 13: The precision comparison results of Example 2 and the original reagents SYPH (Syphilis Treponema pallidum antibody) and HIV (Human Immunodeficiency Virus Type 1+2 Combined Antibody) are as follows:

[0097]

[0098] Table 14: Comparison of the precision of Example 3 and the original supporting reagents MYOG (myoglobin), TNI (troponin), NBNP2 (N-terminal brain natriuretic peptide), and PROG (progesterone)

[0099]

[0100]

[0101] Table 15: The precision comparison results of Example 3 and the original reagents SYPH (Syphilis Treponema pallidum antibody) and HIV (Human Immunodeficiency Virus Type 1+2 Combined Antibody) are as follows:

[0102]

[0103]

[0104] In summary, the substrate solution used in the Ortho VITROS 3600 fully automatic immunoassay system of Examples 1-3 has a good correlation with the precision of the test results of the original reagent, indicating that the self-prepared reagent can still complete the measurement on the Ortho VITROS 3600 fully automatic immunoassay system analyzer on the basis of changing the formula, and the test results are more stable and have better precision.

[0105] Stability test:

[0106] The samples of Example 1 were stored in the dark at 2°C-8°C for 12 months. Stability comparison was performed at the 13th month. The samples were placed in the corresponding positions of the Ortho VITROS 3600 fully automatic immunoassay system for verification testing. Serum from 40 patients was tested. The accuracy and correlation of the results were compared with those of the original reagents. The following table shows the statistical results:

[0107] Table 16: Comparison of the accuracy of the samples after 12 months of storage in Example 1 and the original supporting reagents MYOG (myoglobin), TNI (troponin), and NBNP2 (N-terminal brain natriuretic peptide)

[0108]

[0109]

[0110] Table 17: Comparison of the accuracy of the samples after 12 months of storage in Example 1 with the original supporting reagents PROG (progesterone), SYPH (syphilis antibody), and HIV (human immunodeficiency virus type 1+2 combined antibody)

[0111]

[0112]

[0113] Table 18: Correlation between the test results of the sample after 12 months of storage and the original reagent in Example 1

[0114]

[0115] The results showed that after the self-prepared substrate liquid A and substrate liquid B of Example 1 were stored for 12 months, the test results had a good correlation with the accuracy of the original reagents, and the results were accurate and reliable. In summary, the substrate liquids used in the Orsendo VITROS 3600 fully automatic immunoassay system of Examples 1-3 had a good correlation with the accuracy and precision of the test results of the original reagents after being stored for 12 months, indicating that the self-prepared reagents have better stability than imported reagents based on the change in formula, and the validity period has been increased from 6 months to 12 months, and the results are accurate and reliable.

[0116] The above describes in detail the preferred embodiments of this patent, but this patent is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of this patent.

Claims

1. A substrate solution suitable for a fully automatic immunoassay system, characterized in that: The substrate solution includes solution A and solution B. The components of solution A are: 1.0g / L-3.0g / L of luminol derivative, 10g / L-30g / L of buffer, 0.05g / L-0.5g / L of antibacterial agent, and pH value is 9.00; the components of solution B are: 0.5g / L-2.0g / L of urea peroxide, 0.1g / L-0.5g / L of N-(3-chloro-4-hydroxyphenyl)acetamide, 0.5g / L-3.0g / L of buffer, 0.05g / L-0.5g / L of antibacterial agent, 0.5g / L-5.0g / L of peroxide stabilizer, and pH value is 5.

20.

2. The substrate solution suitable for a fully automatic immunoassay system according to claim 1, characterized in that: In solution A, the luminol derivative is one of luminol, luminol monosodium salt, and isoluminol.

3. The substrate solution suitable for a fully automatic immunoassay system according to claim 1, characterized in that: In solution A, the buffer is one or more of Tris buffer, borax, triethanolamine, imidazole, and 3-amino-1-propanol.

4. The substrate solution suitable for a fully automatic immunoassay system according to claim 1, characterized in that: In liquid A, the antibacterial agent is selected from one or more of ProClin 150, ProClin 200, ProClin 300, and ProClin 950.

5. The substrate solution suitable for a fully automatic immunoassay system according to claim 1, characterized in that: In solution B, the buffer is one or more of formic acid, acetic acid, succinic acid, and citric acid.

6. The substrate solution suitable for a fully automatic immunoassay system according to claim 1, characterized in that: In liquid B, the peroxide stabilizer is one or more of boric acid, borax, 8-hydroxyquinoline, urea, sodium polyacrylate, and disodium ethylenediaminetetraacetic acid.

7. The method for preparing a substrate solution suitable for a fully automatic immunoassay system according to claim 1, comprising the following steps: (1) Preparation of Solution A: Weighing the raw materials: Accurately weigh the luminol derivative, buffer, and antimicrobial agent according to the concentration range described in claim 1; Dissolving: Add the weighed luminol derivative, buffer, and antimicrobial agent to deionized water and stir thoroughly until completely dissolved; Adjusting the pH: Monitor the pH of the solution using a pH meter and adjust the pH to 9.00; Filtration: Filter the solution to remove any insoluble matter and obtain a clear solution A. Aliquot and store: Aliquot the prepared solution A into clean, sealed containers to avoid contamination. (2) Preparation of Solution B: Weighing raw materials: Accurately weigh urea peroxide, N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antibacterial agent and peroxide stabilizer according to the concentration range of claim 1; Dissolve: Dissolve the weighed urea peroxide, Add N-(3-chloro-4-hydroxyphenyl)acetamide, buffer, antimicrobial agent, and peroxide stabilizer to deionized water and stir thoroughly until completely dissolved. Adjust the pH: Monitor the pH of the solution using a pH meter and adjust the pH to 5.

20. Filtration: Filter the solution to remove insoluble matter and obtain clear solution B; Aliquot and store: Aliquot the prepared Solution B into clean, sealed containers to avoid contamination; (4) Storage and use: Store solution A and solution B separately in a dark environment at 2-8°C. When using, according to the operating procedures of the fully automatic immunoassay system, mix solution A and solution B as required or add them separately to the system for testing.

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