Substrate solution formula for antu A2000 full-automatic chemiluminescence immunoassay analyzer and preparation method thereof

Abstract

This invention discloses a substrate solution formulation and preparation method for the Antu A2000 fully automated chemiluminescence immunoassay analyzer, belonging to the field of in vitro diagnostic reagent technology. The substrate solution includes solution A and solution B, wherein solution A consists of hydrogen peroxide, a buffer, and a hydrogen peroxide stabilizer; solution B consists of purified luminol, a luminescence enhancer, and a buffer. The hydrogen peroxide stabilizer is selected from HEDP, 8-hydroxyquinoline, urea, or sodium polyacrylate; the luminescence enhancer is selected from 4-morpholinopyridine, p-iodophenol, or imidazole; luminol is purified by ethanol-water gradient recrystallization and ether extraction. The preparation method includes: weighing and dissolving the components, adjusting the pH, stirring and filtering, and dispensing into opaque containers. This invention uses a stabilizer to improve the stability of hydrogen peroxide, an enhancer to increase the luminescence intensity, and reduces impurity interference, resulting in high sensitivity and low cost. It can completely replace the original reagents, exhibiting significant economic and social benefits.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically, it relates to an improved substrate solution formulation and preparation method for the Antu A2000 fully automated chemiluminescence immunoassay analyzer. Background Technology

[0002] Chemiluminescent immunoassay (CLIA) is a highly mature and advanced technique for detecting trace amounts of bioactive substances. Its origins can be traced back to the late 1970s when scientists began exploring the use of chemiluminescent reactions to label immunoassays as alternatives to traditional radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The development of CLIA benefited from the discovery of chemiluminescent substances such as luminol and acridinium esters, as well as advancements in enzyme catalysis technology. Compared to traditional bioassay techniques, CLIA offers several significant advantages. Firstly, it boasts high sensitivity: CLIA can detect extremely low concentrations of target substances, with detection limits down to the fg / mL (femtograms per milliliter) level. This is 1-2 orders of magnitude lower than the pg / mL (picograms per milliliter) level of ELISA, and even surpasses RIA, which, while highly sensitive, is limited by the decay of radioactive labeling and safety concerns. For example, in detecting trace biomarkers such as alpha-fetoprotein (AFP) or cardiac troponin I (cTnI), CLIA's sensitivity enables early diagnosis. Secondly, it boasts high specificity: CLIA utilizes antigen-antibody specific binding, resulting in an extremely low probability of cross-reactivity and high accuracy. This is due to the direct measurement of the luminescent signal, avoiding background interference that may be introduced by enzyme-catalyzed colorimetry in ELISA. Thirdly, the method is stable and rapid: Labels such as horseradish peroxidase (HRP) are highly stable in the CLIA system, typically reducing detection time to 15-30 minutes, far shorter than the 1-2 hours of ELISA and several hours of RIA, thus meeting the needs of rapid clinical diagnosis. Furthermore, CLIA excels in automation and efficiency: fully automated operation reduces human error, shortening single-test time to within 30 minutes, making it suitable for large-scale sample screening, such as hospital laboratories processing hundreds of samples daily. Finally, it offers a broad detection range: CLIA is applicable to the detection of various biomarkers, covering a wide range from hormones (such as progesterone (PRG) and triiodothyronine (T3)) to tumor markers (such as CA19-9 and AFP) and infectious disease markers (such as HBsAg and tuberculosis-related markers). Finally, it is safe and environmentally friendly: CLIA does not require the use of radioactive materials, avoiding the radiation risks and radioactive waste disposal problems of RIA, and has no toxic residues, meeting modern environmental protection requirements.

[0003] While CLIA inherits all the advantages of RIA, such as high sensitivity and quantitative accuracy, while overcoming the radioactive hazards of RIA and the insufficient sensitivity of ELISA, it is not without its flaws. Compared to ELISA, CLIA's disadvantages include higher equipment and reagent costs, and a shorter shelf life of chemiluminescent substrates. ELISA uses stable chromogenic substrates (such as TMB) with a shelf life of several years, while CLIA's luminescent substrates (such as the luminol-hydrogen peroxide system) are susceptible to light, heat, and pH during storage, leading to decomposition and signal attenuation. Furthermore, CLIA requires a dedicated photometer and automated platform, making its initial investment higher than ELISA; however, in the long run, CLIA's automation efficiency can reduce overall costs. Compared to RIA, CLIA completely eliminates the risk of radioactivity, but early CLIA systems still faced challenges in signal stability, especially in low-concentration samples where the luminescent signal was transient and difficult to capture. In recent years (2020-2025), researchers have improved these problems by introducing nanomaterials and enhancers. For example, the use of metal-organic frameworks (MOFs) can enhance the luminescence intensity of the luminol-hydrogen peroxide system by nearly 2000 times, improving signal persistence and intensity. The core principle of CLIA is to specifically bind the analyte (antigen or antibody) to an antibody or antigen labeled with a chemiluminescent substance (such as luminol, acridinium ester, etc.) to form an immune complex. Subsequently, unbound free labels are removed using a solid-phase support (such as magnetic beads) or plate separation technology. Under specific conditions (such as an alkaline environment, pH 8-10), an oxidant (such as hydrogen peroxide) or a catalyst (such as HRP) is added to excite the label to undergo a redox reaction, generating an excited intermediate. When the intermediate returns to the ground state, it releases photons, generating a light signal (typically at a wavelength of 425 nm). The luminescence intensity is measured using a photomultiplier tube (PMT) or a photometer; this intensity is proportional to the concentration of the analyte and is converted to a concentration value using a standard curve. This process can be divided into direct luminescence (such as the acridinium ester system) and enzyme-catalyzed luminescence (such as the luminol-HRP system). The latter is more common in fully automated instruments because HRP can amplify the signal and improve sensitivity. In recent years, CLIA advancements have included integration with digital microfluidic platforms and smartphone-coupled electrochemiluminescence (ECL) systems. These innovations have enabled CLIA to move from the laboratory to point-of-care testing (POCT).

[0004] The Antu A2000 fully automated chemiluminescence immunoassay analyzer, launched in 2013, is one of Antu Bio's core products and was China's first fully automated CLIA analyzer, later upgraded to the A2000Plus version. This instrument uses a luminol and hydrogen peroxide luminescence system with HRP as the catalyst and is designed for high-throughput laboratory environments. The A2000Plus specifications include: throughput up to 200 tests / h, first result time of only 20 minutes, sample volume range of 5-150 μL, support for continuous loading of 100 sample positions and 24 reagent positions. The instrument measures 1374 x 950 x 1200 mm, operates at 230V, and supports touchscreen operation and digital display. Key features include: an automated sample processing system that automatically completes sample addition, dilution, mixing, and washing, reducing human contamination; a carryover rate of <1 ppm, ensuring accurate results; low reagent consumption, reducing costs; and a built-in quality control module supporting real-time monitoring and data analysis. This instrument holds a significant market position, especially in Asia and emerging markets, where it is popular due to its high cost-effectiveness. According to 2025 data, the Antu A2000 series has been exported to multiple countries. The luminol system is widely studied and applied due to its low cost and mild reaction conditions. Luminol (3-aminophthalohydrazide) was the first luminescent substance used in CLIA and has become an important luminescent reagent since its discovery in the 1950s. In this system, antigens or antibodies label HRP. After the immunization reaction, luminol, as a luminescent substrate, undergoes an oxidation reaction catalyzed by HRP and alkaline hydrogen peroxide: HRP catalyzes the oxidation of luminol by H2O2, generating the excited state of 3-aminophthalate, which releases photons upon decay. The luminescence intensity is proportional to the enzyme concentration, thus indirectly reflecting the content of the target analyte. However, existing CLIA substrate solutions still have several shortcomings, which limit their performance. First, to accommodate the luminescence conditions of luminol in an alkaline environment, the pH of substrate solution A (containing H2O2) is usually set to 4.2, but this acidity is insufficient to completely inhibit the decomposition of H2O2. H₂O₂ readily decomposes spontaneously into water and oxygen in acidic environments, especially under the catalysis of metal ions or photocatalysis, affecting the shelf life of the substrate solution, typically only 12 months. Studies show that H₂O₂ stability is a bottleneck for the shelf life of CLIA reagents, requiring the addition of stabilizers such as HEDP or 8-hydroxyquinoline to chelate metal ions and extend storage time. Secondly, when detecting low concentrations of analytes, the luminescence intensity is insufficient and the signal is short-lived, making it difficult to capture and meet the high sensitivity requirements at the fg / mL level. Traditional luminescence enhancers such as p-iodophenol can increase the intensity, but the increase is limited (usually 2-10 times). Recent advances include the introduction of novel enhancers, such as 4-morpholinopyridine or imidazole, which can further amplify the signal; in addition, the addition of nanomaterials such as gold nanoparticles or MOFs can catalyze the reaction, improving the persistence of glow discharge CLIA and extending the signal stabilization time from several seconds to several minutes.Third, impurities in commercial luminol reagents (such as aniline and phthalic acid) increase baseline noise, produce non-specific luminescence, and reduce the signal-to-noise ratio (S / N). These impurities originate from the synthesis process and their purity is typically only 90-95%. To address this issue, stepwise purification techniques are needed, such as ethanol-water gradient recrystallization combined with activated carbon decolorization and ether extraction, which can improve purity to ≥99.5% and significantly reduce background interference. Fourth, as a high-sensitivity detection device (sensitivity at the fg / mL level), the Antu A2000 urgently needs to be matched with high-performance substrate solutions to fully realize its performance advantages. Currently, while original substrate solutions are reliable, they are expensive and their performance degrades during long-term storage. Research from 2020 to 2025 focuses on optimizing substrate formulations, such as using a Tris-HCl buffer system to stabilize pH and integrating electrochemiluminescence (ECL) to further improve sensitivity. These advances not only improve the practicality of CLIA but also promote its application in food safety and environmental monitoring, such as the detection of pesticide residues or contaminants. In summary, the rapid development of CLIA technology in the field of in vitro diagnostics benefits from its superior performance, but the stability, signal intensity, and purity of the substrate solution remain key challenges. Continuous innovation, such as the introduction of novel stabilizers, enhancers, and purification methods, can further improve the reliability and cost-effectiveness of CLIA systems, providing a more powerful tool for clinical diagnostics. Summary of the Invention

[0005] The problem to be solved To address the problems existing in the prior art, this invention provides a substrate solution formulation and preparation method for the Antu A2000 fully automated chemiluminescence immunoassay analyzer. By introducing hydrogen peroxide stabilizer, luminescence enhancer, and stepwise purification technology of luminol, the stability and luminescence performance of the substrate solution are significantly improved. Specifically, this invention addresses the issue of hydrogen peroxide in substrate solution A easily decomposing under acidic conditions (pH 4.2), leading to a shortened shelf life. It employs stabilizers such as HEDP, 8-hydroxyquinoline, urea, or sodium polyacrylate to chelate metal ions, inhibiting the decomposition reaction and enhancing the stability of hydrogen peroxide, extending the shelf life from 12 months to 18 months. To address the problems of insufficient luminescence intensity and short signal duration in low-concentration samples, enhancers such as 4-morpholinopyridine, p-iodophenol, or imidazole are introduced to increase luminescence intensity by 2-5 times and extend signal duration, improving detection sensitivity to the fg / mL level. To address the issues of high background noise and low signal-to-noise ratio caused by impurities in commercial luminol, a stepwise purification method combining ethanol-water gradient recrystallization with activated carbon decolorization and ether extraction is used to increase luminol purity to ≥99.5%, significantly reducing non-specific luminescence interference. These improvements not only optimize the overall performance of the luminol-hydrogen peroxide luminescence system but also simplify the preparation process, reduce raw material costs, and make the substrate solution more reliable and economical in clinical applications. Technical solution

[0006] To solve the above problems, the present invention adopts the following technical solution.

[0007] A substrate solution suitable for the Antu A2000 fully automated chemiluminescence immunoassay analyzer, comprising solution A and solution B, wherein solution A comprises: 0.5-2 g / L hydrogen peroxide, 1-3 g / L buffer, 0.05-0.1 g / L hydrogen peroxide stabilizer, and pH 4.20; solution B comprises: 0.5-1 g / L purified luminol, 0.05-0.2 g / L luminescence enhancer, 10-20 g / L buffer, and pH 8.90.

[0008] Preferably, the hydrogen peroxide in solution A is commercially available hydrogen peroxide with a content of 30%, which is diluted to obtain a hydrogen peroxide solution with a content of 0.5-2 g / L.

[0009] Preferably, the hydrogen peroxide stabilizer in solution A is one or more of HEDP, 8-hydroxyquinoline, urea, and sodium polyacrylate.

[0010] Preferably, the buffer in solution A is one or more of boric acid, citric acid, tartaric acid, and potassium hydrogen phthalate.

[0011] Preferably, the luminol in solution B is purified by the following method: First, luminol is subjected to gradient recrystallization using an ethanol-water mixed solvent. Industrial-grade luminol is dissolved in hot ethanol at 70°C, activated carbon is added for decolorization, and the solution is filtered to remove insoluble impurities. The filtrate is then slowly cooled to 8°C to precipitate crystals. Second, the obtained crystals are dissolved in water and extracted with an equal volume of diethyl ether. The diethyl ether phase is evaporated to dryness, and the extraction is repeated three times to obtain pure luminol with a purity ≥99.5%.

[0012] Preferably, the luminescence enhancer in liquid B is one or more of 4-morpholinopyridine, p-iodophenol, and imidazole.

[0013] Preferably, the buffer in solution B is one or more of Tris-HCl, borax, potassium dihydrogen phosphate, and glycine-HCl.

[0014] The method for preparing substrate solution suitable for Antu A2000 fully automated chemiluminescence immunoassay analyzer includes the following steps: (1) Preparing substrate solution A: Accurately weigh hydrogen peroxide and buffer according to the concentration range described in claim 1, dissolve in water, monitor with a pH meter and adjust the pH to 4.2, then add hydrogen peroxide stabilizer and stir to dissolve; filter the solution to remove insoluble particles to obtain clear solution A; dispense solution A into opaque sealed containers and store at low temperature; (2) Preparing substrate solution B: According to the concentration range described in claim 1 Accurately weigh the buffer, dissolve it in water, monitor the pH with a pH meter and adjust the pH to 8.9, then add the luminescence enhancer and purified luminol, and stir magnetically for 2 hours under light-protected conditions; filter the solution to remove insoluble particles and obtain clear solution B; dispense solution B into opaque sealed containers and store at low temperature; (3) Storage and use: store substrate solutions A and B in an environment of 2-8℃, and avoid freezing and strong light exposure; when using, add the required amount of substrate solution according to the operating procedures of the fully automated immunoassay system and detect the luminescence intensity.

[0015] Beneficial effects Compared to existing technologies, the advantages of this invention are as follows: This invention comprehensively optimizes the formulation of the luminol luminescent substrate solution. By selecting hydrogen peroxide stabilizers such as 8-hydroxyquinoline, hydrogen peroxide decomposition is effectively inhibited under room temperature conditions, avoiding performance degradation during storage. This significantly extends the shelf life of the substrate solution from 12 months for the original reagent to over 18 months, ensuring long-term stability and reliability. Simultaneously, the introduction of luminescence enhancers such as 4-morpholinopyridine significantly improves luminescence efficacy and signal intensity, ensuring stable luminescence values ​​during low-concentration sample detection. Sensitivity reaches the fg / mL level, far superior to the limited improvement of traditional enhancers, meeting the needs of high-precision clinical diagnosis. Furthermore, through stepwise purification technology of luminol (ethanol-water gradient recrystallization combined with activated carbon decolorization and ether extraction), the purity is increased to ≥99.5%, greatly reducing interference from impurities (such as aniline), effectively reducing background noise, enhancing the signal-to-noise ratio, and improving the accuracy and specificity of detection results. In terms of performance verification, the application of the substrate solution of this invention on the Antu A2000 fully automated chemiluminescence immunoassay analyzer shows that, compared with the original reagent, the accuracy deviation is generally less than 5%, the linear correlation coefficient r>0.99, the precision CV value is between 1.90% and 9.44%, and the stability test shows that it still maintains good correlation after 18 months. These improvements not only enable the substrate solution to comprehensively surpass existing technologies in terms of sensitivity, stability, and reliability, but also simplify the production process, significantly reduce manufacturing costs (raw material costs are reduced by more than 20%), make the product more competitive in price, and significantly reduce the overall cost of clinical testing, alleviating the burden on medical institutions. From a social and economic perspective, this invention promotes the domestic substitution of in vitro diagnostic reagents, drives the sustainable development of the biopharmaceutical industry, and has broad market application prospects. For example, in the detection of infectious diseases (such as tuberculosis and hepatitis B) and tumor markers, it provides a more reliable diagnostic tool, which helps in early intervention and public health management, and is expected to save medical institutions hundreds of millions of yuan in expenditures annually, while improving diagnostic efficiency and benefiting a large number of patients. In summary, this invention, through innovative design, achieves a unity of technological breakthrough and practical value, demonstrating significant industrial driving force and social benefits. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments. Example 1

[0017] The substrate solutions for the Antu Chemiluminescence A2000 fully automated immunoassay system include Substrate Solution A and Substrate Solution B, each containing the following components: Substrate Solution A consists of 1.5 g / L hydrogen peroxide, 2 g / L boric acid, and 0.08 g / L 8-hydroxyquinoline; Substrate Solution B consists of 0.75 g / L purified luminol, 0.1 g / L luminescence enhancer 4-morpholinopyridine, and 15 g / L Tris-HCl buffer. In Example 1, Substrate Solution A prepared at 25℃±0.1℃ has a pH of 4.20 and a conductivity of 6.2 mS / cm. Substrate Solution B at 25℃±0.1℃ has a pH of 8.90 and a conductivity of 11.50 mS / cm.

[0018] The preparation method of substrate solution for the Antu Chemiluminescence A2000 fully automated immunoassay system includes the following steps: (1) Preparation of solution A: According to the concentration range mentioned above, accurately weigh hydrogen peroxide, buffer, and hydrogen peroxide stabilizer, add water to dissolve, and adjust the pH to 4.20; filter to obtain clear solution A; dispense the prepared solution A into clean sealed containers to avoid contamination.

[0019] (2) Preparation of solution B: According to the concentration range mentioned above, accurately weigh the recrystallized and purified luminol, buffer and luminescence enhancer, add water to dissolve, adjust the pH to 8.90, filter, and obtain clear solution B; dispense the prepared solution B into clean sealed containers to avoid contamination.

[0020] (3) Storage and use: Store solution A and solution B in a light-protected environment at 2-8℃. When using, add solution A and solution B to the system as required for testing, according to the operating procedures of the fully automated immunoassay testing system. Example 2

[0021] The substrate solutions used in the Antu Chemiluminescence A2000 fully automated immunoassay system include Substrate Solution A and Substrate Solution B, each containing the following components: Substrate Solution A consists of 1.5 g / L hydrogen peroxide, 2 g / L citric acid, and 0.08 g / L HEDP; Substrate Solution B consists of 0.8 g / L purified luminol, 0.1 g / L imidazole, and 20 g / L borax. In Example 2, Substrate Solution A prepared at 25℃±0.1℃ has a pH of 4.20 and a conductivity of 5.7 mS / cm. Substrate Solution B at 25℃±0.1℃ has a pH of 8.90 and a conductivity of 12.54 mS / cm.

[0022] The preparation method of substrate solution for the Antu Chemiluminescence A2000 fully automated immunoassay system includes the following steps: (1) Preparation of solution A: According to the concentration range, accurately weigh hydrogen peroxide, buffer and hydrogen peroxide stabilizer, add water to dissolve, and adjust the pH to 4.20; filter to obtain clear solution A; dispense the prepared solution A into clean sealed containers to avoid contamination.

[0023] (2) Preparation of solution B: According to the concentration range, accurately weigh the recrystallized and purified luminol, buffer and luminescence enhancer, add water to dissolve, adjust the pH to 8.90, filter, and obtain clear solution B; dispense the prepared solution B into clean sealed containers to avoid contamination.

[0024] (3) Storage and use: Store solution A and solution B in a light-protected environment at 2-8℃. When using, add solution A and solution B to the system as required for testing, according to the operating procedures of the fully automated immunoassay testing system. Example 3

[0025] The substrate solutions used in the Antu Chemiluminescence A2000 fully automated immunoassay system include Substrate Solution A and Substrate Solution B, each containing the following components: Substrate Solution A consists of 2 g / L hydrogen peroxide, 3 g / L tartaric acid, and 0.1 g / L sodium polyacrylate; Substrate Solution B consists of 0.5 g / L purified luminol, 0.05 g / L p-iodophenol, and 20 g / L potassium dihydrogen phosphate. In Example 2, Substrate Solution A prepared at 25℃±0.1℃ has a pH of 4.20 and a conductivity of 6.2 mS / cm. Substrate Solution B at 25℃±0.1℃ has a pH of 8.80 and a conductivity of 11.37 mS / cm.

[0026] The preparation method of substrate solution for the Antu Chemiluminescence A2000 fully automated immunoassay system includes the following steps: (1) Preparation of solution A: According to the concentration range, accurately weigh hydrogen peroxide, buffer and hydrogen peroxide stabilizer, add water to dissolve, and adjust the pH to 4.20; filter to obtain clear solution A; dispense the prepared solution A into clean sealed containers to avoid contamination.

[0027] (2) Preparation of solution B: According to the concentration range, accurately weigh the recrystallized and purified luminol, buffer and luminescence enhancer, add water to dissolve, and adjust the pH to 8.80; filter to obtain clear solution B; dispense the prepared solution B into clean sealed containers to avoid contamination.

[0028] (3) Storage and use: Store solution A and solution B in a light-protected environment at 2-8℃. When using, add solution A and solution B to the system as required for testing, according to the operating procedures of the fully automated immunoassay testing system.

[0029] This invention and the performance test of the substrate solution used in the Antu Chemiluminescence A2000 fully automated immunoassay system: Sensitivity verification: Substrate solutions A and B prepared in Examples 1-3 were placed in corresponding positions on the Antu A2000 fully automated immunoassay system for validation experiments. Serum samples from 40 patients were collected for testing, and the sensitivity and correlation results compared with those obtained using the original reagents are statistically summarized in the table below: Table 1: Comparison of the accuracy of CA19-9 (carbohydrate antigen), PRG (progesterone), and T3 (triiodothyronine) tests between Example 1 and the original reagent.

[0030] Table 2: Comparison of the accuracy of HBsAg (Hepatitis B), AFP (Alpha-fetoprotein), and cTnI (Cardiotropic Troponin) tests between Example 1 and the original reagents.

[0031] Table 3: Correlation between the test results of Example 1 and the original reagent

[0032] The results showed that the detection results of substrate solution A and substrate solution B prepared in Example 1 had a good correlation with the accuracy of the original reagents, and the results were accurate and reliable.

[0033] Table 4: Comparison of accuracy results of CA19-9 (carbohydrate antigen), PRG (progesterone), and T3 (triiodothyronine) tests between Example 2 and the original matching reagents.

[0034] Table 5: Comparison of the accuracy of HBsAg (Hepatitis B), AFP (Alpha-fetoprotein), and cTnI (Calicotinic Acid) tests between Example 2 and the original reagents.

[0035] Table 6: Correlation between the test results of Example 2 and the original reagents

[0036] The results showed that the detection results of substrate solution A and substrate solution B prepared in Example 2 had a good correlation with the accuracy of the original reagents, and the results were accurate and reliable.

[0037] Table 7: Comparison of the accuracy of CA19-9 (carbohydrate antigen), PRG (progesterone), and T3 (triiodothyronine) tests between Example 3 and the original reagent.

[0038] Table 8: Comparison of the accuracy of HBsAg (Hepatitis B), AFP (Alpha-fetoprotein), and cTnI (Cardiotrophin) tests between Example 3 and the original reagents.

[0039] Table 9: Correlation between the test results of Example 1 and the original reagent

[0040] The results showed that the detection results of substrate solutions A and B prepared in Example 3 were well correlated with the accuracy of the original reagents, indicating that the results were accurate and reliable. In summary, the substrate solutions used in Examples 1-3 of the Antu A2000 fully automated immunoassay system showed a good correlation with the accuracy of the original reagents, demonstrating that even with changes to the formulation, the self-prepared reagents can still be used to perform assays on the Antu A2000 fully automated immunoassay system analyzer, and the results are accurate and reliable.

[0041] Precision verification: Take one sample of normal serum and repeat the test 10 times using the self-prepared reagent from Example 1 and the original reagent. Compare the precision of the test results with those obtained using the original reagent.

[0042] Table 10: Precision Comparison Results of Example 1 and Original Matching Reagents for CA19-9 (Carbohydrate Antigen), PRG (Progesterone), and T3 (Triiodinated Thyroid) Tests

[0043] Table 11: Precision Comparison Results of HBsAg (Hepatitis B), AFP (Alpha-fetoprotein), and cTnI (Cardiotropic Troponin) Tests between Example 1 and the Original Matching Reagents

[0044] Table 12: Precision Comparison Results of Example 2 and Original Matching Reagents for CA19-9 (Carbohydrate Antigen), PRG (Progesterone), and T3 (Triiodinated Thyroid) Tests

[0045] Table 13: Precision Comparison Results of HBsAg (Hepatitis B), AFP (Alpha-fetoprotein), and cTnI (Cardiotrophin) Tests between Example 2 and the Original Matching Reagents

[0046] Table 14: Precision Comparison Results of Example 3 and Original Matching Reagents for CA19-9 (Carbohydrate Antigen), PRG (Progesterone), and T3 (Triiodinated Thyroid) Tests

[0047] Table 15: Precision Comparison Results of HBsAg (Hepatitis B), AFP (Alpha-fetoprotein), and cTnI (Cardiotropic Troponin) Tests between Example 3 and the Original Matching Reagents

[0048] In summary, the substrate solutions used in Examples 1-3 of the Antu A2000 fully automated immunoassay system showed a good correlation with the precision of the original reagents, indicating that even with changes to the formulation, the self-prepared reagents can still be used to perform assays on the Antu A2000 fully automated immunoassay system analyzer, and the test results are more stable and have better precision.

[0049] Stability test: Samples from Example 1, stored at 2℃-8℃ in the dark for 18 months, underwent stability comparison at the 19th month. Verification experiments were performed using the corresponding positions on the Antu A2000 fully automated immunoassay system. Serum samples from 40 patients were collected for testing. The accuracy and correlation results compared with those obtained using the original reagents are statistically summarized in the table below: Table 16: Comparison of the accuracy of CA19-9 (carbohydrate antigen), PRG (progesterone), and T3 (triiodothyronine) tests between Example 1 and the original matching reagents.

[0050] Table 17: Comparison of the accuracy of HBsAg (Hepatitis B), AFP (Alpha-fetoprotein), and cTnI (Cardiotrophin) tests between Example 1 and the original reagents.

[0051] Table 18: Correlation between the test results of Example 1 and the original reagent

[0052] The results showed that after 18 months of storage, the substrate solutions A and B prepared in Example 1 exhibited good correlation with the accuracy of the original reagents, indicating that the results were accurate and reliable. In summary, the substrate solutions used in Examples 1-3 of the Antu A2000 fully automated immunoassay system showed good correlation with the accuracy and precision of the original reagents after 18 months of storage, demonstrating that the self-prepared reagents, with modified formulations, exhibited better stability than the original reagents, extending the shelf life from 12 months to 18 months, and providing accurate and reliable results.

[0053] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A substrate solution suitable for the Antu A2000 fully automated chemiluminescence immunoassay analyzer, characterized in that: The substrate solution includes solution A and solution B. Solution A consists of: 0.5-2 g / L hydrogen peroxide, 1-3 g / L buffer, 0.05-0.1 g / L hydrogen peroxide stabilizer, and a pH of 4.

20. Solution B consists of: 0.5-1 g / L purified luminol, 0.05-0.2 g / L luminescence enhancer, and buffer... The concentration of the powder is 10-20 g / L, and the pH value is 8.

90.

2. The substrate solution for the Antu A2000 fully automated chemiluminescence immunoassay analyzer according to claim 1, characterized in that: The hydrogen peroxide in solution A is commercially available hydrogen peroxide with a concentration of 30%, which is diluted to obtain a hydrogen peroxide solution with a concentration of 0.5-2 g / L.

3. The substrate solution for the Antu A2000 fully automated chemiluminescence immunoassay analyzer according to claim 1, characterized in that: The hydrogen peroxide stabilizer in solution A is one or more of HEDP, 8-hydroxyquinoline, urea, and sodium polyacrylate.

4. The substrate solution for the Antu A2000 fully automated chemiluminescence immunoassay analyzer according to claim 1, characterized in that: The buffer in solution A is one or more of boric acid, citric acid, tartaric acid, and potassium hydrogen phthalate.

5. The substrate solution for the Antu A2000 fully automated chemiluminescence immunoassay analyzer according to claim 1, characterized in that: The luminol in solution B was purified as follows: First, luminol was recrystallized in a gradient using an ethanol-water mixed solvent. Industrial-grade luminol was dissolved in hot ethanol at 70°C, activated carbon was added for decolorization, and then the mixture was filtered to remove insoluble impurities. The liquid was slowly cooled to 8°C to precipitate crystals; in the second step, the obtained crystals were dissolved in water and extracted with an equal volume of diethyl ether. The diethyl ether phase was evaporated to dryness, and the extraction was repeated 3 times to obtain pure luminol with a purity ≥99.5%.

6. The substrate solution for the Antu A2000 fully automated chemiluminescence immunoassay analyzer according to claim 1, characterized in that: The luminescence enhancer in solution B is one or more of 4-morpholinopyridine, p-iodophenol, and imidazole.

7. The substrate solution for the Antu A2000 fully automated chemiluminescence immunoassay analyzer according to claim 1, characterized in that: The buffer in solution B is one or more of Tris-HCl, borax, potassium dihydrogen phosphate, and glycine-HCl.

8. The method for preparing the substrate solution suitable for the Antu A2000 fully automated chemiluminescence immunoassay analyzer according to any one of claims 1-7, characterized in that, The steps include: (1) Preparing substrate solution A: Accurately weigh hydrogen peroxide and buffer according to the concentration range described in claim 1, dissolve in water, monitor with a pH meter and adjust the pH to 4.2, then add hydrogen peroxide. Add water stabilizer and stir to dissolve; filter the solution to remove insoluble particles, obtaining clear solution A; dispense solution A into opaque, airtight containers and store at low temperature; (2) Preparation of substrate solution B: Accurately weigh the buffer according to the concentration range described in claim 1, dissolve it in water, monitor the pH using a pH meter and adjust the pH to 8.9, then add the luminescence enhancer and purified luminol. Stir magnetically for 2 hours under light-protected conditions; filter the solution to remove insoluble particles and obtain clear solution B; dispense solution B into opaque sealed containers and store at low temperature; (3) Storage and use: store substrate solution A and solution B in an environment of 2-8℃ and avoid freezing and strong light exposure; when using, add the required amount of substrate solution according to the operating procedures of the fully automated immunoassay system and detect the luminescence intensity.

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