Application of improved isoluminol luminescent substrate in MaglumiX8 chemiluminescence immunoassay system

Abstract

The invention discloses an application of an improved isoluminol luminescent substrate in a MaglumiX8 chemiluminescence immunoassay system, and belongs to the technical field of in vitro diagnostic reagents. The luminescent substrate comprises an alkaline solution containing a metal leaf line complex and a derivative thereof, and a piperazine phenol derivative, and a peroxide solution containing urea and stannate. According to the invention, a metal leaf line substance is added into an alkaline solution, an active structure is stabilized through coordination modification, a reaction with hydrogen peroxide is promoted, a chemiluminescence signal is enhanced, and the luminous efficiency and the detection sensitivity are improved; by adding the piperazine phenol derivative, lasting glow of isoluminol is realized, signal stability is ensured, and result reliability is improved. In a hydrogen peroxide system, a urea / stannate stabilizer is added, so that the stability of a substrate is improved. Experimental verification shows that the substrate has high accuracy correlation with an original reagent, excellent precision and stable performance after being stored at room temperature for 24 months, and is suitable for the detection fields of hormones, tumor markers and the like.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, specifically, it relates to the application of an improved isoluminol luminescent substrate in the MaglumiX8 chemiluminescent immunoassay system. Background Technology

[0002] Chemiluminescence immunoassay (CLIA) is a detection method that combines chemiluminescence technology with the principles of immunoassay. It boasts advantages such as high sensitivity, strong specificity, low background signal, fast detection speed, ease of operation, and high automation, and has been widely applied in clinical diagnostics, environmental monitoring, and food safety. According to market data, the Chinese in vitro diagnostics market has exceeded 70 billion yuan, with a compound annual growth rate of over 18%. CLIA is the fastest-growing sub-sector, with significant potential for import substitution. Common chemiluminescent labels include acridinium esters and their derivatives, luminol and its derivatives (such as isoluminol), and adamantyl dioxane. These labels can bind to enzymes (such as horseradish peroxidase HRP or alkaline phosphatase ALP) or be used directly as luminescent substrates, undergoing a chemical reaction under the action of an oxidant (such as hydrogen peroxide) to generate a light signal that can be captured by a photomultiplier tube.

[0003] The isoluminol chemiluminescence immunoassay system, a core component of CLIA (Collection for International Immunoassay), typically utilizes isoluminol and its derivatives bound to HRP enzymes to produce glow discharge chemiluminescence in the presence of an oxidant, enabling quantitative detection of analytes. This system boasts a high signal-to-noise ratio and a wide linear range (exceeding the 3.0 OD upper limit of ELISA), allowing for highly sensitive detection of trace analytes and rapid signal generation and reading, facilitating rapid diagnosis. The isoluminol chemiluminescence system is widely used in hormone detection, tumor markers, infectious diseases, autoimmune diseases, and myocardial markers. The New Industries Maglumi X8 fully automated chemiluminescence immunoassay analyzer, a high-performance CLIA device, supports throughput up to 600 tests / hour, with first results in just 15 minutes, 24-hour standby, and supports 2800 consecutive tests, making it suitable for medium to large-sized laboratories. However, existing isoluminol luminescence systems have limitations: some systems have short signal durations (glow-type reactions are prone to decay), which may affect the stability of signal readings and require devices with rapid response capabilities; in addition, chemiluminescent reagents such as isoluminol conjugates and hydrogen peroxide are chemically reactive and sensitive to environmental factors such as light and temperature, have poor storage stability (shelf life is usually 6-18 months), and are prone to free radical-induced enzyme inactivation or substrate decomposition, which limits their clinical application efficiency and reliability. Summary of the Invention

[0004] 1. The problem to be solved To address the problems existing in the prior art, this invention provides an improved isoluminol luminescent substrate for use in the MaglumiX8 chemiluminescent immunoassay system. This substrate achieves enhanced luminescence signal, sustained glow, and improved substrate stability by optimizing the metallophylline complex and piperazine-phenol derivative in alkaline solution, as well as the urea / stannate stabilizer in peroxide solution. This improves detection sensitivity, result reliability, and reagent shelf life, making it suitable for in vitro diagnostics fields such as clinical hormones and tumor markers.

[0005] 2. Technical Solution To solve the above problems, the present invention adopts the following technical solution.

[0006] A substrate solution suitable for use with the MaglumiX8 fully automated chemiluminescence immunoassay analyzer for new industries, comprising substrate solution one and substrate solution two. Substrate solution one consists of: an alkaline solution, 1-20 mg / L of luminescence enhancer, 1-20 mmol / L of luminescence stabilizer, 0.1-5 g / L of surfactant, 0.01-0.10 mol / L of buffer, and a pH of 13.0. Substrate solution two consists of: 0.5-2.0 g / L of peroxide, 0.01-0.10 mol / L of buffer, 0.5-5.0 g / L of peroxide stabilizer, and a pH of 5.30.

[0007] Preferably, in substrate solution one, the alkaline solution is a 0.4 mol / L sodium hydroxide aqueous solution.

[0008] Preferably, in substrate solution one, the luminescence enhancer is one of metal chlorophyll, histidine / tryptophan / phenylalanine modified metal chlorophyll, or metal chlorophyll.

[0009] Preferably, in substrate solution one, the luminescent stabilizer is one of piperazine phenol derivatives such as N-methylpiperazine phenol, N-acetylpiperazine phenol, and piperazine phenol hydrochloride.

[0010] Preferably, in substrate solution one, the surfactant is one of sodium dodecyl sulfate, Tween-20, Tween-80, Triton X-100, and Brij-35.

[0011] Preferably, in substrate solution one, the buffer is one or more of carbonate buffer, borate buffer, and glycine.

[0012] Preferably, in substrate solution two, the peroxide is one of urea peroxide, hydrogen peroxide, and sodium perborate.

[0013] Preferably, in substrate solution two, the buffer is one or more of acetic acid, sodium acetate, citric acid, sodium citrate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0014] Preferably, in substrate solution two, the hydrogen peroxide stabilizer is one or more of uric acid, stannate, and organophosphonate.

[0015] As a further technical solution, the substrate solution includes the following components: The substrate solution consists of 0.4 mol / L sodium hydroxide, 5 mg / L histidine-coordinated ferric chloride porphyrin, 10 mmol / L N-acetylpiperazine phenol, 1 g / L Tween-20, and 0.05 mol / L sodium carbonate-sodium bicarbonate buffer.

[0016] The substrate solution consists of two components: 0.5 g / L hydrogen peroxide, 0.05 mol / L acetic acid-sodium acetate buffer, and 2.0 g / L urea.

[0017] Beneficial effects Compared with the prior art, the beneficial effects of the present invention are as follows: This invention introduces metalloporphyrin complexes (such as ferric chloride porphyrin) and their derivatives as luminescence enhancers into an alkaline solution. Through coordination modification (such as histidine coordination), the active conformation of iron ions is stabilized, preventing aggregation and inactivation under alkaline conditions. This promotes the redox reaction with hydrogen peroxide, significantly improving the intensity and efficiency of the chemiluminescence signal, increasing detection sensitivity by over 20%, optimizing the signal-to-noise ratio, and lowering the detection limit to the fg / mL level. Simultaneously, the addition of piperazine-phenol derivatives (such as N-acetylpiperazine-phenol) optimizes reaction kinetics, extending the glow duration to 10-15 minutes, ensuring signal stability, enhancing anti-interference capabilities, reducing background noise by 15%-30%, and significantly improving the reliability of detection results. In the peroxide solution, the addition of stabilizers such as urea / stannate captures reactive oxygen species, blocks the hydrogen peroxide decomposition chain reaction, improves substrate stability, and extends the shelf life at room temperature to over 24 months, superior to the existing 18 months, reducing enzyme inactivation and substrate consumption. Experimental verification shows that this substrate has a high correlation with the original reagent in terms of accuracy (Pearsonr>0.996) for CA19-9, TT3, ALD, HBsAg, HCG, etc., excellent precision (CV<6%, better than the original reagent's 8%), and strong stability. It is suitable for in vitro diagnostics of clinical hormones, tumor markers, etc., reducing costs and promoting import substitution. Attached Figure Description

[0018] Figure 1 This is a graph showing the results of the test run in Example 4.

[0019] Figure 2 This is a graph showing the results of the original reagent CA19-9 (carbohydrate antigen 19-9) on the instrument.

[0020] Figure 3 This is a graph showing the results of the test run in Example 4.

[0021] Figure 4 This is a graph showing the results of the original reagent TT3 (serum total triiodothyronine) administered via instrumentation.

[0022] Figure 5 This is a graph showing the results of the test run in Example 4.

[0023] Figure 6 This is a graph showing the results of using the original reagent ALD (aldosterone) on an instrument.

[0024] Figure 7 This is a graph showing the results of the test run in Example 4.

[0025] Figure 8 This is a graph showing the results of the original HBsAg (hepatitis B surface antigen) reagent.

[0026] Figure 9 This is a graph showing the results of the test run in Example 4.

[0027] Figure 10 This is a graph showing the results of the original HCG (human chorionic gonadotropin) reagent.

[0028] Figure 11 This is a graph showing the precision results of the CA19-9 (Carbohydrate Antigen 19-9) project.

[0029] Figure 12 This is a graph showing the precision results of the TT3 (total triiodothyronine in serum) test.

[0030] Figure 13 This is a graph showing the precision results of the ALD (aldosterone) project.

[0031] Figure 14 This is a graph showing the precision results of the HBsAg (Hepatitis B surface antigen) test.

[0032] Figure 15 This is a graph showing the precision results of the HCG (human chorionic gonadotropin) test.

[0033] Figure 16 The results are a scatter plot and a fitted line showing the correlation between the test results of Example 1 and the original reagent.

[0034] Figure 17 The results are a scatter plot and a fitted line showing the correlation between the test results of Example 2 and the original reagent.

[0035] Figure 18 The results are a scatter plot and a fitted line showing the correlation between the test results of Example 3 and the original reagent.

[0036] Figure 19 The results are a scatter plot and a fitted line showing the correlation between the test results of Example 4 and the original reagent.

[0037] Figure 20 This is a scatter plot and fitted line showing the correlation between the test results of the sample stored for 24 months and the original reagent in Example 4. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments.

[0039] Example 1 The Maglumi X8 fully automated chemiluminescence immunoassay analyzer from New Industries uses two substrate solutions: Substrate Solution 1 and Substrate Solution 2. Substrate Solution 1 consists of 0.4 mol / L sodium hydroxide, 10 mg / L iron phytocyanide, 5 mmol / L N-methylpiperazine phenol, 0.6 g / L sodium dodecyl sulfate, and 0.1 mol / L glycine buffer, with a pH of 13.0. Substrate Solution 2 consists of 0.5 g / L hydrogen peroxide, 2.0 g / L urea, and 0.04 mol / L acetate buffer, with a pH of 5.30. Example 2

[0040] The Maglumi X8 fully automated chemiluminescence immunoassay analyzer from New Industries uses two substrate solutions: Substrate Solution 1 and Substrate Solution 2. Substrate Solution 1 consists of 0.4 mol / L sodium hydroxide, 5 mg / L ferric chloride phytocyanocyanate, 10 mmol / L N-acetylpiperazine phenol, Brij-35 1 g / L, and 0.05 mol / L borate buffer, with a pH of 13.0. Substrate Solution 2 consists of 0.5 g / L hydrogen peroxide, 5.0 g / L urea, and 0.08 mol / L acetate buffer, with a pH of 5.30. Example 3

[0041] The Maglumi X8 fully automated chemiluminescence immunoassay analyzer from New Industries uses two substrate solutions: Substrate Solution 1 and Substrate Solution 2. Substrate Solution 1 consists of 0.4 mol / L sodium hydroxide, 7 mg / L histidine-ferric chloride phytocyanocyanate, 16 mmol / L N-methylpiperazine phenol hydrochloride, Tween-80 4 g / L, and 0.05 mol / L carbonate buffer, with a pH of 13.0. Substrate Solution 2 consists of 0.5 g / L hydrogen peroxide, 10.0 g / L urea, and 0.05 mol / L citrate buffer, with a pH of 5.30. Example 4

[0042] The Maglumi X8 fully automated chemiluminescence immunoassay analyzer from New Industries uses two substrate solutions: Substrate Solution 1 and Substrate Solution 2. Substrate Solution 1 consists of 0.4 mol / L sodium hydroxide, 10 mg / L histidine-ferric chloride phytocyanocyanate, 10 mmol / L N-acetylpiperazine phenol, Tween-20 1 g / L, and 0.05 mol / L borate buffer, with a pH of 13.0. Substrate Solution 2 consists of 0.5 g / L hydrogen peroxide, 5.0 g / L urea, and 0.05 mol / L acetate buffer, with a pH of 5.30.

[0043] This invention was used in performance testing experiments with substrate solutions for the new industry MaglumiX8 fully automated chemiluminescence immunoassay analyzer.

[0044] Accuracy verification The substrate solutions 1 and 2 from Examples 1-4 were used for validation experiments on the New Industries Maglumi X8 fully automated chemiluminescence immunoassay analyzer. Serum samples from 40 patients were collected for the experiments. The accuracy and correlation of the results compared with those from the original reagents are shown in the following table: Table 1: Comparison of the accuracy of CA19-9 (carbohydrate antigen 19-9), TT3 (serum total triiodothyronine), ALD (aldosterone), HBsAg (hepatitis B surface antigen), and HCG (human chorionic gonadotropin) between Example 1 and the original reagents.

[0045] Table 2: Correlation between the test results of Example 1 and the original reagent

[0046] The results show (Table 1, Table 2 and...) Figure 16 The detection results of substrate solution one and substrate solution two in Example 1 showed a good correlation with the accuracy of the original reagent, indicating that the results were accurate and reliable.

[0047] Table 3: Comparison of the accuracy of CA19-9 (carbohydrate antigen 19-9), TT3 (serum total triiodothyronine), ALD (aldosterone), HBsAg (hepatitis B surface antigen), and HCG (human chorionic gonadotropin) between Example 2 and the original reagents.

[0048] Table 4: Correlation between the test results of Example 2 and the original reagent

[0049] The results show (Tables 3 and 4) Figure 17 The detection results of substrate solution one and substrate solution two in Example 2 showed a good correlation with the accuracy of the original reagent, and the results were accurate and reliable.

[0050] Table 5: Comparison of the accuracy of CA19-9 (carbohydrate antigen 19-9), TT3 (serum total triiodothyronine), ALD (aldosterone), HBsAg (hepatitis B surface antigen), and HCG (human chorionic gonadotropin) between Example 3 and the original reagents.

[0051] Table 6: Correlation between the test results of Example 3 and the original reagents

[0052] The results show (Tables 5, 6 and...) Figure 18 The detection results of substrate solution one and substrate solution two in Example 3 showed a good correlation with the accuracy of the original reagent, and the results were accurate and reliable.

[0053] Table 7: Comparison of the accuracy of CA19-9 (carbohydrate antigen 19-9), TT3 (serum total triiodothyronine), ALD (aldosterone), HBsAg (hepatitis B surface antigen), and HCG (human chorionic gonadotropin) tests between Example 4 and the original reagents.

[0054] Table 8: Correlation between the test results of Example 4 and the original reagents

[0055] The results show that ( Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 The detection results of substrate solution one and substrate solution two in Example 4 showed a good correlation with the accuracy of the original reagent, indicating that the results were accurate and reliable.

[0056] Precision verification One serum sample was tested 11 times to compare the detection precision of the original and self-prepared reagents.

[0057] Table 9: Precision Comparison Results of Example 1 and Original Reagents for CA19-9 (Carbohydrate Antigen 19-9), TT3 (Serious Total Triiodothyronine), ALD (Aldosterone), HBsAg (Hepatitis B Surface Antigen), and HCG (Human Chorionic Gonadotropin)

[0058] Table 10: Precision Comparison Results of Example 2 and Original Reagents for CA19-9 (Carbohydrate Antigen 19-9), TT3 (Serious Total Triiodothyronine), ALD (Aldosterone), HBsAg (Hepatitis B Surface Antigen), and HCG (Human Chorionic Gonadotropin)

[0059] Table 11: Precision Comparison Results of Example 3 and Original Reagents for CA19-9 (Carbohydrate Antigen 19-9), TT3 (Serious Total Triiodothyronine), ALD (Aldosterone), HBsAg (Hepatitis B Surface Antigen), and HCG (Human Chorionic Gonadotropin)

[0060] Table 12: Precision Comparison Results of Example 4 with Original Reagents CA19-9 (Carbohydrate Antigen 19-9), TT3 (Serious Total Triiodothyronine), ALD (Aldosterone), HBsAg (Hepatitis B Surface Antigen), and HCG (Human Chorionic Gonadotropin)

[0061] In summary, as shown in Tables 9, 10, 11, and 12, and in conjunction with... Figure 11 , Figure 12 , Figure 13 , Figure 14 and Figure 15 The substrate liquid 1 and substrate liquid 2 in Examples 1-4 showed good precision, and the test results were more stable and more precise than the original.

[0062] Stability test The samples from Example 4 were stored at the same 30°C room temperature environment for 24 months, and their stability was compared. Serum samples from 40 patients were used for testing, and the accuracy and correlation of the results compared with the original reagents are statistically shown in the table below: Table 13: Results of comparing the accuracy of CA19-9 (carbohydrate antigen 19-9), TT3 (total triiodothyronine), ALD (aldosterone), HBsAg (hepatitis B surface antigen), and HCG (human chorionic gonadotropin) tests between the sample preserved for 24 months in Example 4 and the original reagent.

[0063] Table 14: Correlation between test results of Example 4 after 24 months of storage and those of the original reagent

[0064] The results showed (combined with) Figure 19 and Figure 20In Example 4, after substrate 1 and substrate solution 2 were stored at room temperature for 24 months, the test results showed a good correlation with the original reagent, and the results were highly accurate. In summary, after the formulation change in the examples, the stability was better than that of the original reagent, the shelf life increased from 18 months to 24 months, and the test results were highly accurate.

[0065] 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. The application of an improved isoluminol luminescent substrate in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: The luminescent substrate includes substrate solution one and substrate solution two. Substrate solution one consists of an alkaline solution, 1-20 mg / L of luminescence enhancer, 1-20 mmol / L of luminescence stabilizer, 0.1-5 g / L of surfactant, and 0.01-0.10 mol / L of buffer, with a pH of 13.

0. Substrate solution two consists of 0.5-2.0 g / L of peroxide, 0.01-0.10 mol / L of buffer, and 0.5-5.0 g / L of peroxide stabilizer, with a pH of 5.

30.

2. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution one, the alkaline solution is a 0.4 mol / L sodium hydroxide aqueous solution.

3. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution one, the luminescence enhancer is one of metal chlorinated phytophytes, histidine / tryptophan / phenylalanine modified metal phytophytes, or metal chlorinated phytophytes.

4. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution one, the luminescent stabilizer is one of piperazine phenol derivatives such as N-methylpiperazine phenol, N-acetylpiperazine phenol, and piperazine phenol hydrochloride.

5. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution one, the surfactant is one of sodium dodecyl sulfate, Tween-20, Tween-80, Triton X-100, and Brij-35.

6. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution one, the buffer is one or more of carbonate buffer, borate buffer, and glycine.

7. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution two, the peroxide is one of urea peroxide, hydrogen peroxide, and sodium perborate.

8. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution two, the buffer is one or more of acetic acid, sodium acetate, citric acid, sodium citrate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

9. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: In substrate solution two, the peroxide stabilizer is one or more of uric acid, stannate, and organophosphonate.

10. The application of the improved isoluminol luminescent substrate according to claim 1 in the MaglumiX8 chemiluminescent immunoassay system, characterized in that: The first substrate solution consists of 0.4 mol / L sodium hydroxide, 5 mg / L histidine-coordinated ferric chloride phytocyanocyanate, 10 mmol / L N-acetylpiperazine phenol, 1 g / L Tween-20, and 0.05 mol / L sodium carbonate-sodium bicarbonate buffer solution; the second substrate solution consists of 0.5 g / L hydrogen peroxide, 0.05 mol / L acetate-sodium acetate buffer solution, and 2.0 g / L urea.

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