Chemiluminescent kit for detecting pepsinogen II and preparation method thereof

Abstract

The present application relates to a kind of chemiluminescence kit for detecting pepsinogen II, the kit includes enzyme label, luminous substrate liquid and anti-interference buffer system;The anti-interference buffer system includes non-ionic surfactant, animal-derived IgG, antioxidant and catalase;The chemiluminescence kit further includes PG II calibrant and PG II quality control product.The core active ingredient of the enzyme label is β-galactosidase.The enzyme label further includes: β-galactosidase labeled anti-PG II monoclonal antibody, bovine serum albumin, trehalose, sucrose, MgCl2 6H2O, sodium chloride, Proclin 300, phosphate buffer.On the basis of spatial adjacent chemiluminescence analysis method, a brand-new "anti-interference buffer system" is introduced, the resistance to common clinical interferents (such as hemolysis, lipemia, jaundice, heterophilic antibody, etc.) is significantly improved, so that the detection result is more accurate and reliable.

Description

Technical Field

[0001] This invention relates to the field of pepsinogen II detection reagent technology, and in particular to a chemiluminescent reagent kit for detecting pepsinogen II and its preparation method. Background Technology

[0002] Gastric cancer is one of the malignant tumors with consistently high incidence and mortality rates worldwide. Early diagnosis and screening are crucial for improving patient prognosis and increasing five-year survival rates. Among numerous serological screening indicators, pepsinogen (PG) testing plays a central role. Pepsinogen mainly includes two subtypes: pepsinogen I (PG I) and pepsinogen II (PG II). PG I is primarily secreted by the chief cells of the gastric fundic glands, while PG II is jointly secreted by the fundic glands, cardiac glands, pyloric glands, and Brunner's glands in the duodenum. When pathological changes occur in the gastric mucosa, such as in atrophic gastritis, PG I levels decrease significantly, while PG II levels may remain relatively stable or even slightly elevated, resulting in a significant decrease in the PG I / PG II ratio (PGR). Therefore, accurate detection of serum PG II levels, along with PG I concentration and PGR values, constitutes the "gold standard" for assessing gastric mucosal function and screening for atrophic gastritis and gastric cancer risk.

[0003] Currently, in vitro diagnostic methods for PGII mainly include enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). Among them, chemiluminescence immunoassay has become the mainstream choice for clinical laboratories due to its high sensitivity, wide linear range, and high degree of automation. Traditional chemiluminescent immunoassays often employ heterogeneous detection modes, such as magnetic microparticle chemiluminescence. This method involves coating capture antibodies onto magnetic microspheres, forming a complex through antigen-antibody reaction, and then removing free unbound substances through multiple washing steps before finally triggering a chemiluminescent signal. Although the washing steps effectively reduce background interference from the sample matrix and improve detection specificity, they also lead to complex operation procedures, longer detection times, higher reagent costs, and difficulty in achieving truly high throughput and full automation.

[0004] To overcome the aforementioned drawbacks of heterogeneous methods, homogeneous chemiluminescence immunoassay techniques have emerged. Among them, spatial proximity chemiluminescence immunoassay (SPCLA) is a highly promising homogeneous detection technique. Its principle involves using two specific antibodies labeled with an enzyme (such as horseradish peroxidase, HRP) and a luminescent substrate (such as acridinium ester), respectively. When both bind to the same target antigen (such as PGII), the enzyme and substrate are spatially close, resulting in an effective catalytic luminescence reaction. The unbound free label, separated from the antigen, cannot generate an effective signal, resulting in extremely low background. SPCLA technology eliminates the need for a solid-phase support and washing steps, achieving significant simplification of operation and rapid detection (typically completed within 20 minutes), while also significantly reducing reagent costs. Chinese invention patent CN120468424A discloses a kit for detecting PGI based on the SPCLA principle; this technical approach is also applicable to the detection of PGII.

[0005] However, it is precisely this wash-free homogeneous characteristic that exposes an inherent and severe challenge for SPCLA technology when dealing with real clinical samples. Because there is no washing step to remove interfering substances in the reaction system, the complex matrix components in the sample will participate in and affect the immunoreaction and chemiluminescence processes throughout the entire process. These endogenous interfering substances mainly include: 1. Hemoglobin released during hemolysis has pseudoperoxidase activity, which can catalyze nonspecific luminescence of substrates, leading to increased background or false positives. 2. Chylomicrons in lipemia can cause light scattering or non-specific adsorption, quenching luminescence signals or affecting antigen-antibody binding; 3. The high concentration of bilirubin in jaundice, as a strong antioxidant, can quench excited-state photoproducts, leading to signal attenuation; 4. Endogenous interfering factors, such as heterophilic antibodies (especially human anti-mouse antibody, HAMA) and rheumatoid factor (RF), may bridge the capture and detection of antibodies, producing false positive signals.

[0006] For the detection of PG II, the aforementioned matrix effects pose a particularly significant obstacle. Since the baseline concentration of PG II in serum is typically lower than that of PG I, and its concentration variation range is even narrower under certain pathological conditions, higher demands are placed on the accuracy, sensitivity, and anti-interference capabilities of the detection system. Existing SPCLA-based PG II detection protocols often fail to effectively overcome these matrix interferences, leading to significant deviations in results when detecting hemolyzed, lipemic, or jaundiced samples. Recovery rates exceed acceptable clinical ranges (e.g., 85%-115%), severely limiting the application of this technology in routine clinical testing, especially in large-scale population screening.

[0007] Therefore, there is an urgent need in this field for an innovative reagent kit solution that can fundamentally and effectively resist sample matrix interference while retaining all the core advantages of SPCLA technology, such as no washing required, speed, and low cost, and ensure that PGⅡ test results are accurate and reliable in complex real clinical samples. Summary of the Invention

[0008] This invention aims to provide a chemiluminescent reagent kit for detecting pepsinogen II and its preparation method. Based on spatial proximity chemiluminescence analysis, this kit introduces a novel "anti-interference buffer system," significantly improving resistance to common clinical interfering agents (such as hemolysis, lipemia, jaundice, and heterophile antibodies), making the detection results more accurate and reliable. The specific technical solution provided in this application is as follows: In a first aspect, this application provides a chemiluminescent kit for detecting pepsinogen II, the kit comprising an enzyme label, a luminescent substrate solution, and an anti-interference buffer system; the anti-interference buffer system comprising a nonionic surfactant, animal-derived IgG, an antioxidant, and catalase; the chemiluminescent kit further comprising PGII calibrators and PGII quality controls.

[0009] Furthermore, the reagent kit of the present invention has three schemes, as follows: Option 1: The core active ingredient of the enzyme marker is horseradish peroxidase.

[0010] The enzyme markers also include: HRP-labeled anti-PGI monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, Proclin™ 300, and phosphate-buffered saline (PBS).

[0011] Option 2: The core active ingredient of the enzyme marker is alkaline phosphatase.

[0012] The enzyme markers also include: ALP-labeled anti-PGII monoclonal antibody, bovine serum albumin, trehalose, sucrose, MgCl2·6H2O, Proclin™ 300, and Tris-HCl buffer.

[0013] Preferred Option 3: The core active ingredient of the enzyme marker is β-galactosidase.

[0014] The enzyme markers also include: β-galactosidase-labeled anti-PGII monoclonal antibody, bovine serum albumin, trehalose, sucrose, MgCl2·6H2O, sodium chloride, Proclin™ 300, and phosphate buffer.

[0015] Based on the above technical solution, the nonionic surfactant in the anti-interference buffer is Tween-20, the animal-derived IgG is mouse-derived IgG, the antioxidant is ascorbic acid, and the anti-interference buffer also includes PBS; The composition of the anti-interference buffer solution is as follows: Based on a total volume of 1L, each 1L of anti-interference buffer mainly contains Tween-20: 0.1 mL ~ 10 mL; Mouse IgG: 0.1 g ~ 5 g; L-ascorbic acid: 0.01 g ~ 0.5 g; Catalase: 100 U ~ 5000 U.

[0016] Preferably, the mechanism of action of the anti-interference buffer system is as follows: Tween-20: Effectively emulsifies lipid microparticles in lipemic samples, preventing them from non-specifically adsorbing proteins or forming micelles that could affect optical signal transmission.

[0017] Mouse-derived IgG: It effectively neutralizes heterophilic antibodies (such as HAMA) and rheumatoid factor (RF) that may be present in the sample, preventing false positive signals from its bridging marker antibodies.

[0018] Ascorbic acid: As an antioxidant, it reduces the oxidative quenching effect of hemolysin (hemoglobin) and jaundice (bilirubin) substances, and stabilizes chemiluminescent signals.

[0019] Catalase: During the immune response phase, it decomposes the trace amounts of hydrogen peroxide pre-existing in the system, reducing the background. After the addition of a trigger (containing a large amount of hydrogen peroxide), its activity is instantly saturated, thus allowing the normal luminescence reaction to proceed. This dynamic balance effectively overcomes the interference of pseudoperoxidases such as hemolysates.

[0020] More preferably, the anti-interference buffer system is independently packaged.

[0021] More preferably, the anti-interference buffer system also includes a specific HAMA blocking agent.

[0022] Secondly, the present invention also provides a method for preparing a chemiluminescent reagent kit for detecting pepsinogen II, the preparation method of which includes the following steps: S1: Preparation of enzyme-labeled antibody: 0.8 g / L β-galactosidase-labeled anti-PGII monoclonal antibody, 60.0 g / L trehalose, 25.0 g / L sucrose, 10.0 g / L bovine serum albumin, 5.0 g / L sodium chloride, 0.5 g / L magnesium chloride, and 1.0 mL / L Proclin™ 300 preservative were dissolved and brought to a final volume in 0.01 M, pH 7.2 phosphate buffer. After mixing, the mixture was filtered sterilized and aliquoted for storage. S2: Preparation of the luminescent substrate solution: Preparation of luminescent label: Prepare approximately 800 mL of 0.01 M PBS buffer, pH 7.2; dissolve casein, trehalose, and sucrose sequentially with stirring; add dihydroacrylidine-labeled anti-PGII monoclonal antibody and mix gently; add Proclin™ 300 and mix well; bring the volume to 1.0 L with PBS buffer (pH 7.2); filter sterilize using a 0.22 μm filter membrane; aliquot and store at 2–8 °C protected from light; Trigger preparation: Dissolve 0.15 mM Galacton-Star chemiluminescent substrate, 20.0 g / L Tris-HCl, and 5.0 g / L sodium chloride in purified water, adjust the pH to 8.8-9.2, mix thoroughly in the dark, and then make up to volume and dispense. S3: Preparation of the anti-interference buffer system: Dissolve 3.0 mL / L Tween-20, 2.5 g / L mouse IgG, 0.2 g / L L-ascorbic acid, 500 U / L catalase, 0.5 g / L specific HAMA blocker and 1.0 mL / L Proclin™ 300 preservative in Tris-HCl buffer at pH 7.2-7.4, mix well, filter sterilize, and aliquot for storage; S4: Preparation of calibrators and quality control samples: Dilute pepsinogen II antigen to a series of concentrations of 0.78-50 ng / mL using protein-containing matrix buffer, and aliquot to prepare calibrators; and prepare low, medium and high concentration quality control samples in a similar manner.

[0023] Thirdly, based on the same invention, this invention also provides a method for using a chemiluminescent reagent kit for detecting pepsinogen II, the steps of which are as follows: S1. Pre-incubation: Take 10 μL of the clinical sample to be tested and mix it with an equal volume of anti-interference buffer system, and incubate for 5 minutes; S2. Immunological reaction: Add β-Gal enzyme label and dihydroacrylamide luminescent label, incubate for 10-15 minutes to form a double antibody sandwich complex of "enzyme-labeled antibody-PGⅡ antigen-luminescent labeled antibody"; S3, Triggered luminescence: After injecting a trigger, the substrate is catalyzed by β-Gal on the complex, and after hydrolysis, it generates a chemiluminescent signal through a series of reactions. S4. Signal Detection: The instrument measures the luminous intensity (RLU). S5. Result Calculation: The PGI concentration is automatically calculated based on the calibration curve.

[0024] The present invention has the following beneficial effects: Compared with existing technologies, the chemiluminescent reagent kit for detecting pepsinogen II (PGII) and its preparation method provided by the present invention have the following significant advantages: 1. Strong resistance to matrix interference, accurate and reliable test results. This invention creatively introduces a unique anti-interference buffer system into a homogeneous chemiluminescence detection system for PGII. This system, through the synergistic action of four components—a nonionic surfactant, animal-derived IgG, an antioxidant, and catalase—effectively neutralizes and inhibits major interfering sources such as lipemia, heterophilic antibodies / rheumatoid factor, hemolysis / jaundice, and endogenous peroxidase. This makes the kit exhibit extremely strong robustness when dealing with complex clinical samples, with a stable recovery rate within the clinically required range of 85%-115%. It effectively solves the industry problem of PGII being highly susceptible to matrix interference in homogeneous immunoassays due to its wash-free nature, and its detection accuracy is comparable to that of traditional heterogeneous magnetic microparticle chemiluminescence methods.

[0025] 2. It combines the ease of operation of homogeneous methods with the detection accuracy of heterogeneous methods. This invention perfectly retains the core advantages of spatial proximity chemiluminescence analysis (SPCLA): ease of operation, no cleaning required, fast reaction speed (typically completed within 20 minutes), and low cost. Simultaneously, through breakthroughs in anti-interference technology, it successfully achieves detection accuracy and reliability approaching or even reaching the level of heterogeneous methods, providing clinical laboratories with a high-quality solution for PGII detection that is both fast and accurate, particularly suitable for high-throughput and emergency testing scenarios.

[0026] 3. Achieving high sensitivity and high signal-to-noise ratio tailored to the characteristics of PGⅡ detection. In particular, when using a β-galactosidase (β-Gal) luminescent system, the system achieves extremely low background signal because endogenous β-galactosidase activity is almost non-existent in human serum. Combined with an optimized label formulation, the PGⅡ kit provided by this invention has a lower limit of detection and a higher signal-to-noise ratio, enabling accurate detection of low-concentration PGⅡ samples. This provides crucial technical support for accurately calculating the PGR ratio and early detection of gastric mucosal atrophy.

[0027] 4. The platform is highly versatile, facilitating industrialization and clinical application. The proposed anti-interference buffer system is highly versatile, perfectly compatible with mainstream chemiluminescence systems such as horseradish peroxidase (HRP)-acrididine ester, alkaline phosphatase (ALP)-AMPPD / CSPD, and β-galactosidase (β-Gal)-Galacton-Star. This flexibility allows the technology to be widely applied to different chemiluminescence immunoassay platforms and instruments without altering the core anti-interference strategy, significantly expanding its industrialization potential and market application scope.

[0028] 5. The reagent has good stability, extending shelf life and reducing costs. This invention optimizes the stabilizer formulation of enzyme markers based on the characteristics of different enzyme systems. For example, in the ALP and β-Gal system, by adding Mg²⁺... + Essential cofactors are included, and a trehalose-sucrose binary stabilization system is employed to maximize enzyme activity. An effective combination of stabilizers is also incorporated into the HRP system. These measures, combined with the inherent high stability of the ALP and β-Gal systems, significantly extend the shelf life of the kit (typically 12-18 months at 2-8°C), reducing storage and logistics costs.

[0029] 6. Excellent detection performance indicators The PGⅡ reagent kit provided by this invention has the following excellent comprehensive performance indicators: High sensitivity: The lower limit of detection (LoD) is significantly lower than the clinical decision level, which can meet the needs of early screening for low-value detection.

[0030] Wide linear range: It can cover the entire clinical testing range from normal to abnormally high values, and most samples do not require manual dilution, reducing operational errors.

[0031] High correlation: Compared with approved mainstream commercially available PGⅡ detection kits (such as magnetic microparticle chemiluminescence assay), the correlation coefficient (R²) is greater than 0.97, indicating high consistency of results.

[0032] In summary, this invention, through ingenious anti-interference buffer system design and system integration, successfully overcomes the key technical bottlenecks of homogeneous chemiluminescence immunoassay in PGII detection, providing a perfect solution for PGII detection that is highly anti-interference, accurate, reliable, rapid, simple, stable, and economical. This solution has significant clinical value and market potential for promoting the large-scale application of homogeneous immunoassay technology in gastric function assessment and early gastric cancer screening. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the present invention can be obtained commercially. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0034] Anti-PGII monoclonal antibody was purchased from Nanjing Jingda Biotechnology Co., Ltd., and PGII antigen was purchased from Nanjing Ainodi Biotechnology Co., Ltd.

[0035] Example 1: Chemiluminescent reagent kit for HRP (horseradish peroxidase) system A chemiluminescent reagent kit for detecting pepsinogen II, characterized in that the kit comprises an enzyme label, a luminescent substrate solution, and an anti-interference buffer system; the anti-interference buffer system comprises a nonionic surfactant, animal-derived IgG, an antioxidant, and catalase; the chemiluminescent reagent kit further comprises PGII calibrators and PGII quality controls. The core active ingredient of the enzyme label is horseradish peroxidase. The luminescent substrate solution comprises a luminescent label, a luminescent auxiliary agent, and a trigger.

[0036] 1.1 Enzyme markers The enzyme markers also include: HRP-labeled anti-PGII monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, Proclin™ 300, and phosphate-buffered saline (PBS).

[0037] Taking the preparation of 1L of working solution as an example, the components are as follows: Enzyme markers: 1.2 g of HRP-labeled anti-PGⅡ monoclonal antibody, 10.0 g of BSA, 25.0 g of trehalose, 10.0 g of sucrose, and 1.0 mL of Proclin™ 300 were diluted to 1.0 L with phosphate-buffered saline PBS (0.01 M, pH 7.4).

[0038] Among them, HRP-labeled anti-PGII monoclonal antibody is the core active ingredient, used to catalyze the luminescent reaction; BSA is a carrier protein that can reduce adsorption and stabilize the antibody; trehalose is an advanced stabilizer that prevents inactivation due to high temperature and freeze-thaw cycles; sucrose is an auxiliary stabilizer that works synergistically with trehalose; Proclin™ 300 is a preservative used to inhibit microbial growth; and PBS is used to provide a stable pH environment.

[0039] 1.2 Luminescent substrate liquid 1.2.1 Luminescent markers: 0.6 g of 9,10-dihydroacridine-labeled anti-PGⅡ monoclonal antibody, 5.0 g of casein, 20.0 g of trehalose, 10.0 g of sucrose, and 1.0 mL of Proclin™ 300 were diluted to 1.0 L with phosphate-buffered saline PBS (0.01 M, pH 7.4).

[0040] Among them, the 9,10-dihydroacrylidine-labeled anti-PGII monoclonal antibody is the core luminescent material, which emits photons after oxidation; casein is a stabilizer used to reduce non-specific adsorption; trehalose is a protectant used to stabilize the labeled antibody; sucrose is also a protectant, working synergistically with trehalose; Proclin™ 300 is a preservative; and PBS is the buffer system.

[0041] 1.2.2 Luminescent auxiliary agent: 1.82 g citric acid, 10.45 g sodium citrate, 0.2 mL 30% hydrogen peroxide solution, and purified water to a final volume of 1 L.

[0042] The preparation method is as follows: dissolve citric acid and sodium citrate in water, adjust the pH to 5.0-6.0, and finally add 30% hydrogen peroxide solution and mix well.

[0043] The function of the luminescent auxiliary is to provide hydrogen peroxide (H2O2), a substrate necessary for the HRP catalytic reaction.

[0044] 1.2.3 Trigger: Tris-HCl 6.06 g, sodium chloride (NaCl) 9.0 g, concentrated HCl (36-38%) 2.1 mL, Tween-20 1.0 mL, and purified water to a final volume of 1.0 L.

[0045] Concentrated hydrochloric acid is used to fine-tune the pH, while Tween-20 is used to promote mixing and clean the pipeline. The triggering agent functions to provide an alkaline environment, initiating and enhancing the chemiluminescent reaction of acridine esters.

[0046] 1.3 Anti-interference buffer system: Tris-HCl 5.0 g, sodium chloride 8.0 g, Tween-20 2.0 mL, mouse IgG 1.0 g, L-ascorbic acid 0.1 g, catalase 1000 U, Proclin™ 300 1.0 mL, and purified water to a final volume of 1.0 L.

[0047] Among them, Tris-HCl is the basis of the buffer system; sodium chloride is used to maintain an isotonic environment; Tween-20 is used to neutralize lipemia interference and reduce adsorption; murine IgG is used to neutralize heterophilic antibodies and rheumatoid factor; L-ascorbic acid is an antioxidant used to resist the quenching effect of hemolysis and jaundice; catalase is used to decompose background hydrogen peroxide, reduce background levels, and overcome the interference of endogenous peroxidase.

[0048] Preparation method: Dissolve the above ingredients in water, adjust the pH to 7.2-7.4, filter to sterilize, dispense into containers, and store at 2-8℃.

[0049] Example 2: Chemiluminescent reagent kit for ALP (alkaline phosphatase) system A chemiluminescent reagent kit for detecting pepsinogen II, characterized in that the kit comprises an enzyme label, a luminescent substrate solution, and an anti-interference buffer system; the anti-interference buffer system comprises a nonionic surfactant, animal-derived IgG, an antioxidant, and catalase; the chemiluminescent reagent kit further comprises PGII calibrators and PGII quality controls. The core active component of the enzyme label is alkaline phosphatase (ALP). The luminescent substrate solution comprises the luminescent label and a trigger.

[0050] 2.1 Enzyme markers The enzyme markers also include: ALP-labeled anti-PGII monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, magnesium chloride (MgCl2·6H2O), Proclin™ 300, and Tris-HCl buffer.

[0051] Taking the preparation of 1L of working solution as an example, the components are as follows: Enzyme markers: 1.0 g of ALP-labeled anti-PGⅡ monoclonal antibody, 10.0 g of BSA, 40.0 g of trehalose, 15.0 g of sucrose, 1.0 g of magnesium chloride (MgCl2·6H2O), and 1.0 mL of Proclin™ 300 were diluted to 1.0 L with Tris-HCl buffer (0.05 M pH 8.0).

[0052] Among them, the ALP-labeled anti-PGII monoclonal antibody is the core active ingredient, used to catalyze the luminescent reaction; BSA is a carrier protein that can reduce adsorption and stabilize the antibody; trehalose is an excellent stabilizer that provides low-temperature protection; sucrose is an auxiliary stabilizer that works synergistically with trehalose; magnesium chloride is an essential cofactor for ALP that can maintain enzyme activity; Proclin™ 300 is a preservative used to inhibit microbial growth; and Tris-HCl is used to provide the optimal alkaline environment for ALP.

[0053] 2.2 Luminescent substrate liquid 2.2.1 Luminescent markers: 0.6 g of 9,10-dihydroacrylidine-labeled anti-PGⅡ monoclonal antibody, 5.0 g of casein, 20.0 g of trehalose, 10.0 g of sucrose, and 1.0 mL of Proclin™ 300 were brought to a final volume of 1.0 L with Tris-HCl buffer (0.05 M pH 8.0).

[0054] Among them, the 9,10-dihydroacrylidine-labeled anti-PGII monoclonal antibody is the core luminescent material, which emits photons after oxidation; casein is a stabilizer used to reduce non-specific adsorption; trehalose is a protectant used to stabilize the labeled antibody; sucrose is also a protectant, working synergistically with trehalose; Proclin™ 300 is a preservative; and Tris-HCl is the buffer system.

[0055] 2.2.2 Trigger: CSPD 0.2 mM, Emerald-II™ luminescence enhancer 0.5% (v / v), Tris-HCl 20.0 g, NaCl 5.0 g, purified water to a final volume of 1.0 L.

[0056] Preparation method: Dissolve the above components in water and adjust the pH to 9.8. This reagent must be prepared and stored away from light.

[0057] This trigger is used to provide the ALP chemiluminescent substrate and the high pH environment required for chemiluminescence, thereby initiating and enhancing the chemiluminescent reaction of acridine esters.

[0058] 2.3 Anti-interference buffer system: Tris-HCl 5.0 g, sodium chloride 8.0 g, Tween-20 2.5 mL, mouse IgG 1.8 g, L-ascorbic acid 0.12 g, catalase 1500 U, Proclin™ 300 1.0 mL, and purified water to a final volume of 1.0 L.

[0059] Among them, Tris-HCl is the basis of the buffer system; sodium chloride is used to maintain an isotonic environment; Tween-20 is used to neutralize lipemia interference and reduce adsorption; murine IgG is used to neutralize heterophilic antibodies and rheumatoid factor; L-ascorbic acid is an antioxidant used to resist the quenching effect of hemolysis and jaundice; catalase is used to decompose background hydrogen peroxide, reduce background levels, and overcome the interference of endogenous peroxidase.

[0060] Preparation method: Dissolve the above ingredients in water, adjust the pH to 7.2-7.4, filter to sterilize, dispense into containers, and store at 2-8℃.

[0061] 2.4 Reagent Kit Testing Procedure S1: Pre-incubation: Take 10 μL of the clinical sample to be tested and mix it with an equal volume of anti-interference buffer system, and incubate for 5 minutes; S2: Immunological reaction: Add ALP enzyme marker and acridinium ester luminescent marker, incubate for 10-15 minutes to form a double antibody sandwich complex of "enzyme-labeled antibody-PGII antigen-luminescently labeled antibody"; S3: Triggered luminescence: After injecting a trigger, the substrate is catalyzed by ALP on the complex, generating a chemiluminescent signal; S4: Signal detection: The instrument measures the luminous intensity (RLU); S5: Result Calculation: The PGⅡ concentration is automatically calculated based on the calibration curve.

[0062] Example 3: Chemiluminescent kit for the β-Gal (β-galactosidase) system A chemiluminescent reagent kit for detecting pepsinogen II, characterized in that the kit comprises an enzyme label, a luminescent substrate solution, and an anti-interference buffer system; the anti-interference buffer system comprises a nonionic surfactant, animal-derived IgG, an antioxidant, and catalase; the chemiluminescent reagent kit further comprises PGII calibrators and PGII quality controls. The core active ingredient of the enzyme label is β-galactosidase. The luminescent substrate solution comprises the luminescent label and a trigger.

[0063] The principle of using β-galactosidase is as follows: since there is almost no endogenous β-galactosidase activity in human serum, this system can achieve extremely low background signal, thereby obtaining extremely high signal-to-noise ratio and detection sensitivity.

[0064] 3.1 Enzyme markers The enzyme markers also include: β-galactosidase-labeled anti-PGI monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, magnesium chloride (MgCl2·6H2O), sodium chloride, Proclin™ 300, and phosphate-buffered saline (PBS).

[0065] Taking the preparation of 1L of working solution as an example, the components are as follows: Enzyme markers: 0.8 g of β-galactosidase-labeled anti-PGII monoclonal antibody, 10.0 g of BSA, 60.0 g of trehalose, 25.0 g of sucrose, 0.5 g of magnesium chloride (MgCl2·6H2O), 5.0 g of sodium chloride, and 1.0 mL of Proclin™ 300 were added and brought to a final volume of 1.0 L with phosphate-buffered saline (PBS 0.01 M, pH 7.2).

[0066] The core active ingredient is the β-Gal-labeled anti-PGI monoclonal antibody, which catalyzes the luminescent reaction. BSA is a carrier protein that reduces adsorption and stabilizes the antibody. Trehalose is the primary stabilizer, providing excellent protection for β-Gal. Sucrose is a secondary stabilizer that works synergistically with trehalose. Magnesium chloride is used to stabilize enzyme activity, as some β-Gal isoenzymes require magnesium ions. Sodium chloride is used to maintain ionic strength. Proclin™ 300 is a preservative. PBS provides a neutral and optimal storage environment.

[0067] The preparation method of enzyme markers is as follows: 1. Prepare approximately 800 mL of 0.01M PBS buffer, pH 7.2.

[0068] 2. Dissolve sodium chloride, magnesium chloride, trehalose, sucrose, and BSA in sequence while stirring.

[0069] 3. Add β-Gal-labeled anti-PGI monoclonal antibody and mix gently.

[0070] 4. Add Proclin™ 300 and mix well.

[0071] 5. Adjust the volume to 1.0 L with PBS buffer (pH 7.2).

[0072] 6. Use a 0.22 μm filter membrane for filtration and sterilization.

[0073] 7. Pack into smaller portions and store at 2-8℃ away from light.

[0074] 3.2 Luminescent substrate solution: 3.2.1 Luminescent markers 0.6 g of 9,10-dihydroacridine-labeled anti-PGⅡ monoclonal antibody, 5.0 g of casein, 20.0 g of trehalose, 10.0 g of sucrose, and 1.0 mL of Proclin™ 300 were diluted to 1.0 L with phosphate-buffered saline PBS (0.01 M, pH 7.4).

[0075] Among them, the 9,10-dihydroacrylidine-labeled anti-PGII monoclonal antibody is the core luminescent material, which emits photons after oxidation; casein is a stabilizer used to reduce non-specific adsorption; trehalose is a protectant used to stabilize the labeled antibody; sucrose is also a protectant, working synergistically with trehalose; Proclin™ 300 is a preservative; and PBS is the buffer system.

[0076] 3.2.2 Trigger Galacton-Star 0.15 mM, Tris-HCl 20.0 g, sodium chloride 5.0 g, and purified water to a final volume of 1 L.

[0077] Among them, Galacton-Star is the substrate of β-Gal, which is catalyzed to generate a luminescent intermediate. Tris-HCl provides the high pH environment (~pH 9.0) required to trigger luminescence, initiates and enhances the chemiluminescent reaction of acridine ester, and sodium chloride is used to maintain ionic strength.

[0078] The preparation method is as follows: 1. Dissolve Tris and NaCl in approximately 900 mL of purified water and stir until dissolved.

[0079] 2. Adjust the pH to 8.8-9.2 using concentrated HCl.

[0080] 3. Add the Galacton-Star substrate and stir gently until completely dissolved.

[0081] 4. Make up to 1.0 L with purified water.

[0082] 5. Handle with complete protection from light. Use brown bottles or opaque containers to dispense the contents and store at 2-8℃ away from light.

[0083] 3.3 Anti-interference buffer system: Tris-HCl 5.0 g, sodium chloride 8.0 g, Tween-20 3.0 mL, mouse IgG 2.5 g, L-ascorbic acid 0.2 g, catalase 500 U, specific HAMA blocker 0.5 g, Proclin™ 300 1.0 mL, purified water to a final volume of 1.0 L.

[0084] Among them, Tris-HCl is a buffer pair; sodium chloride is used to maintain an isotonic environment; Tween-20 is used to neutralize lipemia interference and reduce adsorption; murine IgG is used to neutralize heterophilic antibodies; L-ascorbic acid is an auxiliary antioxidant; catalase is still retained as a preventive measure to combat the very few samples containing highly active catalase, further ensuring signal stability; the specific HAMA blocker forms a double blockade with murine IgG to ensure absolute safety.

[0085] Preparation method: 1. Dissolve Tris and NaCl in water and stir until dissolved.

[0086] 2. Add Tween-20, mouse IgG, ascorbic acid, and catalase in sequence, ensuring that each component is dissolved before adding the next.

[0087] 3. Adjust the pH to 7.2-7.4.

[0088] 4. Add Proclin™ 300 and mix well.

[0089] 5. Add purified water to a final volume of 1.0 L, filter to sterilize, dispense, and store at 2-8℃.

[0090] The chemiluminescent reagent kit for the β-Gal system benefits from the near absence of endogenous β-Gal activity in human serum, resulting in extremely low background signal, a very high signal-to-noise ratio (S / N), and enormous sensitivity potential. It exhibits excellent anti-interference properties; combined with an anti-interference buffer system, it has a strong neutralizing ability against interference from lipemia, heterophilic antibodies, etc., and its tolerance to hemolysis and jaundice is also superior to the HRP system. It demonstrates high stability; β-galactosidase is a very stable enzyme, and its reagents can maintain activity for extended periods at 2-8°C. It also boasts high safety; its substrates, such as Galacton-Star, are safer and more stable than some HRP substrates.

[0091] 3.4 Reagent Kit Testing Procedure: S1: Pre-incubation: Take 10 μL of the clinical sample to be tested and mix it with an equal volume of anti-interference buffer system, and incubate for 5 minutes.

[0092] S2: Immunological reaction: Add β-Gal enzyme marker and dihydroacrylamide luminescent marker, incubate for 10-15 minutes to form a double antibody sandwich complex of "enzyme-labeled antibody-PGⅡ antigen-luminescent labeled antibody".

[0093] S3: Triggered luminescence: After injecting a trigger, the substrate is catalyzed by β-Gal on the complex, and after hydrolysis, it generates a chemiluminescent signal through a series of reactions.

[0094] S4: Signal detection: The instrument measures the luminous intensity (RLU).

[0095] S5: Result Calculation: The PGI concentration is automatically calculated based on the calibration curve.

[0096] Experiment Example 1: Sensitivity vs. Signal-to-Noise Ratio (S / N) Comparison Experiment This experiment aims to quantitatively evaluate and compare the blank background, limit of detection (LoD), and signal-to-noise ratio of three kits: Example 1 (HRP), Example 2 (ALP), and Example 3 (β-Gal), thereby assessing their sensitivity.

[0097] Experimental Materials and Methods: Test kits: Example 1 (HRP), Example 2 (ALP), Example 3 (β-Gal) PGII chemiluminescence kit.

[0098] Blank sample: matrix buffer without PGII (same as calibrator matrix).

[0099] Low concentration samples: Prepare a PGII antigen solution with a concentration of 0.8 ng / mL using the matrix buffer described above.

[0100] Instrument: Chemiluminescence immunoassay analyzer Experimental methods: Blank samples were tested using each kit, with 20 repeated measurements. Low-concentration samples were tested using each kit, with 10 repeated measurements. The luminescence value (RLU) for each test was recorded.

[0101] Calculate the blank mean, limit of detection (LoD), low concentration sample mean, and signal-to-noise ratio (S / N) respectively.

[0102] The experimental data are shown in Table 1: Table 1

[0103] Analysis of the experimental data in Table 1 shows that... Regarding background noise and limit of detection (LOD), the background noise of Example 3 (β-Gal) (885 RLU) was significantly lower than that of Example 1 (4,520 RLU) and Example 2 (2,150 RLU). This directly verifies the "naturally low background" advantage resulting from the near absence of endogenous β-galactosidase activity in human serum. Simultaneously, Example 3 exhibited the smallest background fluctuation (SD=42), indicating its system is highly stable. Therefore, the calculated LOD of Example 3 (0.04 ng / mL) is far superior to the other two, demonstrating its ability to detect lower concentrations of PGII and its highest sensitivity.

[0104] In terms of signal-to-noise ratio (SNR), it is the gold standard for measuring detection reliability. A higher SNR indicates that the effective signal is more easily distinguished from background noise, resulting in higher reliability and accuracy. Example 3 (β-Gal) achieved a high SNR of 54.8, which is 2.3 times that of Example 2 (24.3) and 4.2 times that of Example 1 (12.9). This means that when detecting a low concentration of PGII (0.8 ng / mL), Example 3 produced an effective signal intensity 54.8 times that of its background noise, exhibiting the highest reliability and accuracy with minimal data fluctuation.

[0105] In conclusion, this experiment, through scientific comparative data, fully demonstrates that Example 3 (the β-galactosidase-based kit) exhibits the lowest detection limit and the highest signal-to-noise ratio when detecting PGII, with its sensitivity indicators being optimal across the board. This makes it the preferred solution for clinical applications requiring high sensitivity and accuracy.

[0106] Experimental Example 2: Robustness Testing in Complex Real-World Clinical Samples This experimental example aims to verify the consistency between the embodiments of the present invention and the reference method (gold standard) in real clinical samples with varying degrees of endogenous interfering substances (hemolysis, lipemia, jaundice, heterophilic antibodies), thereby assessing the accuracy and robustness of its detection results.

[0107] Experimental Materials and Methods: Test kits: Example 1 (HRP), Example 2 (ALP), Example 3 (β-Gal) PGII kits.

[0108] Reference method: A commercially available magnetic microparticle chemiluminescence PGII detection kit with approved and recognized performance. This method is a heterogeneous detection method, and the washing step effectively removes most matrix interferences. The results are used as the benchmark for accuracy assessment in this experiment.

[0109] Samples: Fifty residual serum samples were collected from clinical patients, covering low, medium, and high PGII concentration ranges and including varying degrees of hemolysis (H), lipemia (L), and jaundice (I). A sample with an apparent normal appearance (N) was also included as a control.

[0110] Experimental methods: All 50 samples were tested in duplicate using three different test kits and a reference method, and the average value was taken. The relative deviation of each sample under different implementation methods was calculated based on the results of the reference method.

[0111] Relative deviation (%) = [(Measured value of example - Measured value of reference method) / Measured value of reference method] × 100% The experimental data are shown in Table 2: Table 2: Test results and relative deviation of 50 clinical samples

[0112] Table 3: Statistical Analysis Results of Relative Deviation under Different Sample Types

[0113] Analysis of the experimental data in Tables 2 and 3 shows that: Regarding overall accuracy, as shown in Table 3, Example 3 (β-Gal) had the closest mean relative deviation to 0 (+0.5%) across all 50 samples, and the smallest mean absolute deviation (MARD) (2.2%). This indicates that its measured values ​​were most consistent with the true values ​​of the reference method, and its overall accuracy was the best.

[0114] In terms of anti-interference capability analysis, For hemolyzed samples: Hemoglobin has pseudoperoxidase activity, which severely interferes with the HRP system. Example 1 showed a significant positive deviation (+16.8%). Example 3, thanks to its optimized anti-interference buffer system (high concentration of ascorbic acid and catalase) and β-Gal enzyme that does not react with hemoglobin, kept the deviation at an extremely low level of +1.2%.

[0115] For jaundice samples: bilirubin is a known luminescence quencher. Example 1 showed a significant negative bias (-13.5%), indicating that its luminescence signal was severely quenched. Example 3, with its high concentration of antioxidant (ascorbic acid) and extremely low background signal, effectively resisted this quenching effect, with a bias of only -1.1%.

[0116] For lipemia samples: chylomicrons can cause light scattering and non-specific adsorption. All three examples were affected to some extent, but Example 3, containing the highest concentration of Tween-20, showed the best emulsification effect on lipids, with the deviation controlled at -0.9%.

[0117] For seemingly normal samples: even in samples without visible interference, the consistency of Example 3 (+0.4%) was significantly better than the other two, demonstrating the superior accuracy of its underlying detection system.

[0118] Overall robustness ranking: Example 3 (β-Gal) > Example 2 (ALP) > Example 1 (HRP).

[0119] In summary, this experiment, using a large number of complex real clinical samples, fully demonstrates that Example 3 (the β-galactosidase-based kit) exhibits the highest consistency with the reference method, the smallest deviation, and the greatest stability across various types of interfering samples. Its powerful anti-interference buffer system, combined with its stable enzyme system, endows this kit with unparalleled robustness, ensuring accurate and reliable results in complex real clinical testing environments, fundamentally solving the industry challenge of homogeneous immunoassay being susceptible to matrix interference.

[0120] Comparative Example 1: Performance test without anti-interference buffer system This comparative example aims to verify that, without the addition of the anti-interference buffer system described in this invention, the detection accuracy of the kits of Example 1 (HRP), Example 2 (ALP), and Example 3 (β-Gal) for complex clinical samples is significantly reduced, thus demonstrating that the anti-interference buffer system is a universal and key improvement of this invention.

[0121] Experimental Materials and Methods Test group: Kits 1, 2, and 3 of Examples, but without the accompanying anti-interference buffer system. During testing, the samples were mixed and pre-incubated with an equal volume of plain PBS buffer (pH 7.4).

[0122] Control group: Complete kits of Examples 1, 2, and 3 (using the matching anti-interference buffer system).

[0123] Reference method: Approved commercially available magnetic microparticle chemiluminescence PGII detection kit.

[0124] Sample: 15 samples with very obvious interference were specifically selected from the sample library of Experiment Example 2: Five samples with obvious hemolysis, five samples with obvious jaundice, and five samples with obvious lipemia. Experimental methods: All 15 samples were tested simultaneously using the respective test groups, control groups, and reference methods. The relative deviations between the test groups and control groups were calculated based on the results obtained using the reference method.

[0125] Relative deviation (%) = [(Measured value - Reference method measured value) / Reference method measured value] × 100% The experimental data are shown in Table 4: Table 4: Performance Comparison of No and Added Anti-interference Buffer System

[0126] Analysis of the experimental data in Table 4 shows that: Regarding the universality and criticality of the anti-interference buffer system: regardless of whether HRP, ALP, or β-Gal systems are used, removing the anti-interference buffer system drastically increases the detection bias of all kits on interfering samples, making it completely unacceptable (the average absolute value of the bias far exceeds the clinically acceptable standard of 10%). This demonstrates that the anti-interference buffer system has a universal and crucial improving effect on the entire homogeneous chemiluminescence detection platform, and is the core of this invention.

[0127] Regarding the analysis of performance degradation: Example 1 (HRP) showed the most severe degradation: it was most affected by endogenous substances, and after losing protection, the deviation was over ±30%. Example 2 (ALP), although improved, was still unacceptable: without an anti-interference system, the average absolute value of the deviation still exceeded 12%, proving its insufficient anti-interference ability. Example 3 (β-Gal) completely lost its "inherent advantage": this is a very convincing finding. Without an anti-interference system, the deviation of the β-Gal system in hemolysis and jaundice even exceeded that of the ALP system with system protection. This especially proves that the buffer system is not just icing on the cake, but a necessary condition to ensure that the β-Gal system achieves its theoretical optimal performance. Its performance degradation factor was the highest (22-fold), which shows that the optimal enzyme system and the optimal anti-interference technology are a "powerful combination" and neither can be dispensed with.

[0128] Regarding the demonstration of performance recovery: After adding an anti-interference buffer system (control group), the accuracy of all systems was greatly and decisively improved, and the deviation was controlled at an excellent level.

[0129] Among them, Example 3 (β-Gal) achieved near-perfect accuracy (average deviation <1%) after being combined with the anti-interference system, highlighting the powerful synergistic effect of combining the "optimal enzyme system" and the "universal anti-interference technology".

[0130] This comparative example, through comparative experiments, confirms that the anti-interference buffer system designed in this invention is a general, crucial, and indispensable technical means to overcome the problem of matrix interference in homogeneous chemiluminescence detection. It not only significantly improves the practicality of the HRP and ALP systems but is also a prerequisite for unleashing the full potential of the β-galactosidase system, making it the optimal solution. Therefore, Example 3 (β-Gal) represents the best combination of the "anti-interference buffer system" and the homogeneous chemiluminescence detection system.

[0131] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A chemiluminescent reagent kit for detecting pepsinogen II, characterized in that, The kit includes an enzyme label, a luminescent substrate solution, and an anti-interference buffer system; the anti-interference buffer system contains a nonionic surfactant, animal-derived IgG, an antioxidant, and catalase; the chemiluminescent kit also includes PGII calibrators and PGII quality controls.

2. The chemiluminescent reagent kit for detecting pepsinogen II according to claim 1, characterized in that, The core active ingredient of the enzyme marker is alkaline phosphatase.

3. A chemiluminescent reagent kit for detecting pepsinogen II according to claim 2, characterized in that, The enzyme markers also include: ALP-labeled anti-PGII monoclonal antibody, bovine serum albumin, trehalose, sucrose, MgCl2·6H2O, Proclin™ 300, and Tris-HCl buffer.

4. A chemiluminescent reagent kit for detecting pepsinogen II according to claim 1, characterized in that, The core active ingredient of the enzyme marker is β-galactosidase.

5. A chemiluminescent reagent kit for detecting pepsinogen II according to claim 4, characterized in that, The enzyme markers also include: β-galactosidase-labeled anti-PGII monoclonal antibody, bovine serum albumin, trehalose, sucrose, MgCl2·6H2O, sodium chloride, Proclin™ 300, and phosphate buffer.

6. A chemiluminescent reagent kit for detecting pepsinogen II according to claim 1, characterized in that, The nonionic surfactant in the anti-interference buffer is Tween-20, the animal-derived IgG is mouse-derived IgG, the antioxidant is ascorbic acid, and the anti-interference buffer also includes PBS; The composition of the anti-interference buffer solution is as follows: Based on a total volume of 1L, each 1L of anti-interference buffer mainly contains Tween-20: 0.1 mL ~ 10 mL; Mouse IgG: 0.1 g ~ 5 g; L-Ascorbic acid: 0.01 g ~ 0.5 g; Catalase: 100 U ~ 5000 U.

7. A chemiluminescent reagent kit for detecting pepsinogen II according to claim 6, characterized in that, The anti-interference buffer system also includes a specific HAMA blocking agent.

8. A chemiluminescent reagent kit for detecting pepsinogen II according to claim 1, characterized in that, The anti-interference buffer system is independently packaged.

9. A method for preparing a chemiluminescent reagent kit for detecting pepsinogen II, characterized in that, The preparation method of the reagent kit is as follows: S1: Preparation of enzyme-labeled antibody: 0.8 g / L β-galactosidase-labeled anti-PGII monoclonal antibody, 60.0 g / L trehalose, 25.0 g / L sucrose, 10.0 g / L bovine serum albumin, 5.0 g / L sodium chloride, 0.5 g / L magnesium chloride, and 1.0 mL / L Proclin™ 300 preservative were dissolved and brought to a final volume in 0.01 M, pH 7.2 phosphate buffer. After mixing, the mixture was filtered for sterilization and aliquoted for storage. S2: Preparation of the luminescent substrate solution: Preparation of luminescent label: Prepare approximately 800 mL of 0.01 M PBS buffer, pH 7.2; dissolve casein, trehalose, and sucrose sequentially with stirring; add dihydroacrylidine-labeled anti-PGII monoclonal antibody and mix gently; add Proclin™ 300 and mix well; bring the volume to 1.0 L with PBS buffer (pH 7.2); filter sterilize using a 0.22 μm filter membrane; aliquot and store at 2–8 °C protected from light; Trigger preparation: Dissolve 0.15 mM Galacton-Star chemiluminescent substrate, 20.0 g / L Tris-HCl, and 5.0 g / L sodium chloride in purified water, adjust the pH to 8.8-9.2, mix thoroughly in the dark, and then make up to volume and dispense. S3: Preparation of the anti-interference buffer system: Dissolve 3.0 mL / L Tween-20, 2.5 g / L mouse IgG, 0.2 g / L L-ascorbic acid, 500 U / L catalase, 0.5 g / L specific HAMA blocker and 1.0 mL / L Proclin™ 300 preservative in Tris-HCl buffer at pH 7.2-7.4, mix well, filter sterilize, and aliquot for storage; S4: Preparation of calibrators and quality control samples: Dilute pepsinogen II antigen to a series of concentrations of 0.78-50 ng / mL using protein-containing matrix buffer, and aliquot to prepare calibrators; and prepare low, medium and high concentration quality control samples in a similar manner.

10. A method of using a chemiluminescent reagent kit for detecting pepsinogen II, characterized in that, The usage steps are as follows: S1. Pre-incubation: Take 10 μL of the clinical sample to be tested and mix it with an equal volume of anti-interference buffer system, and incubate for 5 minutes; S2. Immunological reaction: Add β-Gal enzyme label and dihydroacrylamide luminescent label, incubate for 10-15 minutes to form a double antibody sandwich complex of "enzyme-labeled antibody-PGII antigen-luminescent labeled antibody"; S3, Triggered luminescence: After injecting a trigger, the substrate is catalyzed by β-Gal on the complex, and after hydrolysis, it generates a chemiluminescent signal through a series of reactions. S4. Signal Detection: The instrument measures the luminous intensity (RLU). S5. Result Calculation: The PGI concentration is automatically calculated based on the calibration curve.

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