Chemiluminescent kit for detecting pepsinogen I and preparation method thereof

Abstract

The present application relates to a kind of chemiluminescence kit for detecting pepsinogen I, including 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 also includes PGI calibrator and PGI quality control product.The core active ingredient of the enzyme label is beta-galactosidase.The enzyme label also includes: beta-galactosidase labeled anti-PGI monoclonal antibody, bovine serum albumin, trehalose, sucrose, magnesium chloride, sodium chloride, Proclin 300, PBS.Based on the advantages of spatial proximity method luminescence analysis method, by introducing a brand-new "anti-interference buffer system", the resistance ability to clinical common sample mechanism effect (such as hemolysis, lipemia, jaundice, heterophilic antibody, etc.) is significantly improved, so that detection result is more accurate and reliable in complex real clinical sample.

Description

Technical Field

[0001] This invention relates to the field of pepsinogen I detection reagents, and particularly to a chemiluminescent reagent kit for detecting pepsinogen I and its preparation method. Background Technology

[0002] Gastric cancer is a common malignant tumor of the digestive tract, and its early diagnosis is crucial for improving patient survival. Serum pepsinogen I (PGI) levels and its ratio (PGR) to pepsinogen II (PGII) are important serological indicators reflecting gastric mucosal function and screening for atrophic gastritis and gastric cancer risk. Therefore, developing highly sensitive and accurate PGI detection kits has significant clinical implications.

[0003] Currently, the main methods for detecting PGⅠ include enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLIA). Among them, chemiluminescence immunoassay has become the mainstream technology due to its high sensitivity, good specificity, and wide linear range. Traditional chemiluminescent immunoassays often employ heterogeneous detection modes, such as magnetic microparticle chemiluminescence, which relies on the capture of antigen-antibody complexes by a solid-phase support (such as magnetic microspheres) and the separation of free and bound labels through a washing step. Although this method effectively reduces background interference, the washing step leads to complex operation procedures, long detection times, and high reagent costs.

[0004] To simplify operation and improve efficiency, homogeneous chemiluminescence immunoassay (CLIA) technology has emerged. Among them, spatial proximity chemiluminescence assay (SPCLA) is an advanced homogeneous detection technique. Its principle involves using two specific antibodies, one labeled with an enzyme (e.g., horseradish peroxidase, HRP) and the other with a luminescent substrate (e.g., acridil ester). When these antibodies bind to the same target antigen to form a complex, the enzyme and substrate are spatially close, catalyzing an effective chemiluminescent reaction. Unbound free labels, separated from each other, cannot react effectively, resulting in a very low background signal. This method eliminates the need for a solid-phase support and washing steps, achieving significant simplification of operation and rapid detection, and substantially reducing reagent costs. Chinese invention patent CN120468424A discloses a kit for detecting PGI based on the SPCLA principle.

[0005] However, this technology presents an inherent major challenge: due to its wash-free homogeneous nature, complex matrix components in the sample (such as hemoglobin released from hemolysis, chylomicrons in lipemia, bilirubin in jaundice, and endogenous interfering substances like heterophilic antibodies and rheumatoid factor) remain in the reaction system throughout the entire process and cannot be removed by washing. These substances can non-specifically interfere with antigen-antibody binding, quench chemiluminescence signals, or generate false positive signals, leading to significant deviations in test results. This high sensitivity to sample matrix effects severely limits the accuracy and reliability of SPCLA technology in the detection of complex clinical samples, restricting its widespread application.

[0006] Therefore, there is an urgent need in this field for an improved kit that can effectively overcome sample matrix interference and significantly improve the accuracy and robustness of detection results while retaining all the advantages of SPCLA technology, such as no washing required, speed, and low cost. Summary of the Invention

[0007] This invention aims to provide an improved kit for detecting pepsinogen I and its preparation method. Based on the advantages of spatial proximity luminescence analysis, this kit introduces a novel "anti-interference buffer system" to significantly enhance its resistance to common clinical sample mechanism effects (such as hemolysis, lipemia, jaundice, heterophilic antibodies, etc.), making the detection results more accurate and reliable in complex real clinical samples.

[0008] Specifically, the technical solution provided in this application is as follows: In a first aspect, this application provides a chemiluminescent kit for detecting pepsinogen I, 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 PGI calibrators and PGI 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-PGI monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, magnesium chloride (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-PGI monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, magnesium chloride (MgCl2·6H2O), sodium chloride, Proclin™ 300, and phosphate-buffered saline (PBS).

[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; 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] Secondly, the present invention also provides a method for preparing a chemiluminescent reagent kit for detecting pepsinogen I, the preparation method of which includes the following steps: S1: Preparation of enzyme-labeled antibody: 1.0 g / L of β-galactosidase-labeled anti-PGI monoclonal antibody, 50.0 g / L of trehalose, 20.0 g / L of sucrose, 10.0 g / L of bovine serum albumin, 0.5 g / L of magnesium chloride and 1.0 mL / L of 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 to remove bacteria, aliquoted and stored. 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-PGI 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 0.1-0.3 mM Galacton-Star chemiluminescent substrate, 20.0 g / L tris(hydroxymethyl)aminomethane, 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 2.0 mL / L Tween-20, 1.5 g / L mouse IgG, 0.05 g / L L-ascorbic acid, 500 U / L catalase 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 I antigen to a series of concentrations of 1.56-200 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.

[0022] The present invention has the following beneficial effects: Compared with existing technologies, the chemiluminescent reagent kit for detecting pepsinogen I (PGI) and its preparation method provided by the present invention have the following significant advantages: 1. Strong resistance to matrix interference, resulting in accurate and reliable results. This invention innovatively introduces a unique anti-interference buffer system into a homogeneous chemiluminescence detection system. This system utilizes a multi-functional synergistic mechanism involving four components: nonionic surfactants, animal-derived IgG, antioxidants, and catalase. These components effectively neutralize and inhibit major interfering sources such as lipemia, heterophilic antibodies, hemolysis / jaundice, and endogenous peroxidase. This results in exceptional robustness when dealing with complex real-world clinical samples, with a stable recovery rate within the clinically required range of 85%-115%. It fundamentally solves the matrix effect problem caused by the wash-free nature of homogeneous immunoassay technology, and its detection accuracy is comparable to traditional heterogeneous methods (such as magnetic particle chemiluminescence).

[0023] 2. It combines the simplicity of homogeneous methods with the accuracy of heterogeneous methods. This invention perfectly retains the core advantages of spatial proximity chemiluminescence analysis (SPCLA), namely, ease of operation, no cleaning required, fast reaction speed, and low cost. Simultaneously, through breakthroughs in anti-interference technology, it achieves the detection accuracy and reliability of near-heterogeneous methods, providing clinical laboratories with a high-efficiency and reliable high-quality detection solution.

[0024] 3. Excellent detection performance indicators High sensitivity: Low detection limit (LoD) effectively distinguishes low-concentration samples from blank background.

[0025] Wide linear range: It can cover clinical testing needs from normal to abnormally high values, eliminating the need for manual dilution and reducing operational errors.

[0026] High correlation: The results show high consistency with mainstream and recognized diagnostic kits on the market.

[0027] 4. 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. Detailed Implementation

[0028] 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.

[0029] Anti-PGⅠ monoclonal antibody was purchased from Nanjing Jingda Biotechnology Co., Ltd., and PGⅠ antigen was purchased from Nanjing Ainodi Biotechnology Co., Ltd.

[0030] Example 1: Chemiluminescent reagent kit for HRP (horseradish peroxidase) system A chemiluminescent reagent kit for detecting pepsinogen I, comprising 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 also includes PGI calibrators and PGI 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.

[0031] 1.1 Enzyme markers The enzyme markers also include: HRP-labeled anti-PGI monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, Proclin™ 300, and phosphate-buffered saline (PBS).

[0032] Taking the preparation of 1L of working solution as an example, the components are as follows: Enzyme markers: 1.0 g of HRP-labeled anti-PGI monoclonal antibody, 10.0 g of BSA, 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 phosphate-buffered saline (PBS).

[0033] Among them, HRP-labeled anti-PGI 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.

[0034] 1.2 Luminescent substrate liquid 1.2.1 Luminescent markers: 0.5 g of 9,10-dihydroacrylidine-labeled anti-PGI 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 added and brought to a final volume of 1.0 L with phosphate-buffered saline (PBS).

[0035] Among them, the 9,10-dihydroacrylidine-labeled anti-PGI 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.

[0036] 1.2.2 Luminescent auxiliary agent: Citric acid 1.82 g, sodium citrate 10.45 g, 30% hydrogen peroxide solution 0.3 mL, purified water to a final volume of 1 L.

[0037] 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.

[0038] The function of the luminescent auxiliary is to provide hydrogen peroxide (H2O2), a substrate necessary for the HRP catalytic reaction.

[0039] 1.2.3 Trigger: Tris-HCl 6.06 g, sodium chloride (NaCl) 9.0 g, concentrated HCl (36-38%) 2.1 mL, Tween-20 2.0 mL, and purified water to a final volume of 1.0 L.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Preparation method: Dissolve the above components in water, adjust the pH to 7.2-7.4, filter to sterilize, dispense into containers, and store at 2-8℃.

[0044] Example 2: Chemiluminescent reagent kit for ALP (alkaline phosphatase) system A chemiluminescent reagent kit for detecting pepsinogen I, 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 PGI calibrators and PGI 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.

[0045] 2.1 Enzyme markers The enzyme markers also include: ALP-labeled anti-PGI monoclonal antibody, bovine serum albumin (BSA), trehalose, sucrose, magnesium chloride (MgCl2·6H2O), Proclin™ 300, and Tris-HCl buffer.

[0046] 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-PGI monoclonal antibody, 10.0 g of BSA, 50.0 g of trehalose, 20.0 g of sucrose, 1.0 g of magnesium chloride (MgCl2·6H2O), and 1.0 mL of Proclin™ 300 were brought to a final volume of 1.0 L with Tris-HCl buffer.

[0047] Among them, the ALP-labeled anti-PGI 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.

[0048] 2.2 Luminescent substrate liquid 2.2.1 Luminescent markers: 0.5 g of 9,10-dihydroacrylidine-labeled anti-PGI 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 added and brought to a final volume of 1.0 L with Tris-HCl buffer.

[0049] Among them, the 9,10-dihydroacrylidine-labeled anti-PGI 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.

[0050] 2.2.2 Trigger: CSPD 0.25 mM, Tris-HCl 20.0 g, NaCl 5.0 g, purified water to a final volume of 1.0 L.

[0051] Preparation method: Dissolve the above components in water and adjust the pH to 9.5-10.0. This reagent must be prepared and stored away from light.

[0052] 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.

[0053] 2.3 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.

[0054] 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.

[0055] Preparation method: Dissolve the above components in water, adjust the pH to 7.2-7.4, filter to sterilize, dispense into containers, and store at 2-8℃.

[0056] 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-PGI 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 PGI concentration is automatically calculated based on the calibration curve.

[0057] Example 3: Chemiluminescent kit for the β-Gal (β-galactosidase) system A chemiluminescent reagent kit for detecting pepsinogen I, 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 PGI calibrators and PGI quality controls. The core active ingredient of the enzyme label is β-galactosidase. The luminescent substrate solution comprises a luminescent label and a trigger.

[0058] 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.

[0059] 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).

[0060] Taking the preparation of 1L of working solution as an example, the components are as follows: Enzyme markers: 1.0 g of β-galactosidase-labeled anti-PGI monoclonal antibody, 10.0 g of BSA, 50.0 g of trehalose, 20.0 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 0.01 M phosphate-buffered saline (PBS) at pH 7.2.

[0061] 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.

[0062] The preparation method of enzyme markers is as follows: 1. Prepare approximately 800 mL of 0.01M PBS buffer, pH 7.2.

[0063] 2. Dissolve sodium chloride, magnesium chloride, trehalose, sucrose, and BSA in sequence while stirring.

[0064] 3. Add β-Gal-labeled anti-PGI monoclonal antibody and mix gently.

[0065] 4. Add Proclin™ 300 and mix well.

[0066] 5. Adjust the volume to 1.0 L with PBS buffer (pH 7.2).

[0067] 6. Use a 0.22 μm filter membrane for filtration and sterilization.

[0068] 7. Pack into smaller portions and store at 2-8℃ away from light.

[0069] 3.2 Luminescent substrate solution: 3.2.1 Luminescent markers 0.5 g of 9,10-dihydroacrylidine-labeled anti-PGI 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 added and brought to a final volume of 1.0 L with phosphate-buffered saline (PBS).

[0070] Among them, the 9,10-dihydroacrylidine-labeled anti-PGI 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.

[0071] 3.2.2 Trigger Galacton-Star 0.3mM, Tris-HCl 20.0g, sodium chloride 5.0g, purified water to a final volume of 1L.

[0072] 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.

[0073] The preparation method is as follows: 1. Dissolve Tris and NaCl in approximately 900 mL of purified water and stir until dissolved.

[0074] 2. Adjust the pH to 8.8-9.2 using concentrated HCl.

[0075] 3. Add the Galacton-Star substrate and stir gently until completely dissolved.

[0076] 4. Make up to 1.0 L with purified water.

[0077] 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.

[0078] 3.3 Anti-interference buffer system: Tris-HCl 5.0 g, sodium chloride 8.0 g, Tween-20 2.0 mL, mouse IgG 1.5 g, L-ascorbic acid 0.05 g, catalase 500 U, Proclin™ 300 1.0 mL, and purified water to a final volume of 1.0 L.

[0079] 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; mouse IgG is used to neutralize heterophilic antibodies; L-ascorbic acid is an auxiliary antioxidant to further ensure signal stability; catalase is still retained as a preventive measure to combat the very few samples containing highly active catalase.

[0080] Preparation method: 1. Dissolve Tris and NaCl in water and stir until dissolved.

[0081] 2. Add Tween-20, mouse IgG, ascorbic acid, and catalase in sequence, ensuring that each component is dissolved before adding the next.

[0082] 3. Adjust the pH to 7.2-7.4.

[0083] 4. Add Proclin™ 300 and mix well.

[0084] 5. Add purified water to a final volume of 1.0 L, filter to sterilize, dispense, and store at 2-8℃.

[0085] 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.

[0086] 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.

[0087] S2: Immunoreaction: 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-PGI antigen-luminescent labeled antibody".

[0088] 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.

[0089] S4: Signal detection: The instrument measures the luminous intensity (RLU).

[0090] S5: Result Calculation: The PGI concentration is automatically calculated based on the calibration curve.

[0091] Experiment Example 1: Sensitivity vs. Signal-to-Noise Ratio (S / N) Comparison Experiment This experiment aims to quantitatively 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 evaluating their sensitivity.

[0092] Experimental Materials and Methods Kits: Examples 1 (HRP), 2 (ALP), and 3 (β-Gal) were prepared using the same anti-PGI monoclonal antibody and the same batch of PGI antigen.

[0093] Blank sample: PGI zero-concentration calibrator. Low-concentration sample: PGI sample with a concentration of 1.0 ng / mL.

[0094] Instrument: Chemiluminescence immunoassay analyzer Experimental method: Blank samples were tested 20 times and low-concentration samples were tested 10 times using each kit. The luminescence value (RLU) of each test was recorded.

[0095] Calculate the blank mean, limit of detection (LoD), low concentration sample mean, and signal-to-noise ratio (S / N) respectively.

[0096] The experimental data are shown in Table 1, with units of ng / mL. Table 1

[0097] Analysis of the experimental data in Table 1 shows that, regarding blank background and limit of detection, at the concentration level, the average concentration of the blank sample in Example 3 (β-Gal) (0.02 ng / mL) and the calculated limit of detection (0.03 ng / mL) are significantly lower than those of the other two examples. This demonstrates that the β-Gal system, due to its extremely low endogenous background, can distinguish trace amounts of target analytes from noise.

[0098] Regarding the signal-to-noise ratio (SNR), Example 3 (β-Gal) had a significantly higher SNR (48.0) than Example 2 (19.6) and Example 1 (13.1). This indicates that when detecting low-concentration samples of 1.0 ng / mL, the effective signal intensity of the β-Gal system is 48 times that of its background noise, resulting in the highest reliability and accuracy of the detection results with minimal data fluctuation.

[0099] In terms of accuracy, all systems showed mean detection values ​​close to the true value for 1.0 ng / mL samples, but β-Gal achieved this with lower background and higher signal-to-noise ratio, which is even more valuable.

[0100] This experimental example, through scientific comparison at the concentration level, fully demonstrates that Example 3 (a kit based on β-galactosidase) is optimal in all key sensitivity indicators. It has the lowest detection limit and the highest signal-to-noise ratio, making it the preferred solution for clinical applications requiring high sensitivity and accuracy.

[0101] 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 in real clinical samples with varying degrees of endogenous interfering substances, thereby assessing the accuracy and robustness of its detection results.

[0102] Experimental Materials and Methods Test kits: Example 1 (HRP), Example 2 (ALP), Example 3 (β-Gal). Reference method: Approved commercially available magnetic microparticle chemiluminescence PGI detection kits (as the standard for accuracy assessment).

[0103] Samples: Fifty residual serum samples from clinical patients were collected, covering different concentration ranges (low, medium, and high) and containing varying degrees of hemolysis, lipemia, and jaundice.

[0104] Experimental methods: All samples were tested simultaneously using three different test kits and a reference method. The relative deviation of each sample under different implementation methods was calculated based on the results obtained using the reference method.

[0105] Relative deviation (%) = [(Measured value of example - Measured value of reference method) / Measured value of reference method] × 100%.

[0106] Experimental data are shown in Tables 2 and 3, where the concentration unit is ng / mL, and the sample type is N for normal, H for hemolysis, L for lipemia, and I for jaundice.

[0107] Table 2

[0108] The statistical analysis results obtained from the experimental data in Table 2 are shown in Table 3: Table 3

[0109] The experimental data above show that the kit in Example 3 has the highest consistency with the reference method in various types of clinical samples, with the smallest deviation and the most stable results.

[0110] Comparative Example 1: Performance test without adding an anti-interference buffer system This comparative example aims to verify that, without the addition of an anti-interference buffer system, the detection accuracy of the kits in 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.

[0111] Materials and Methods Test group: Kits from Examples 1, 2, and 3, but without the accompanying anti-interference buffer system. During testing, the samples were mixed with an equal volume of PBS buffer.

[0112] Control group: Complete kits from Examples 1, 2, and 3 (using the matching anti-interference buffer system) Reference method: Approved commercially available magnetic microparticle chemiluminescence PGI detection kit.

[0113] Samples: 15 samples with significant interference were specifically selected (as before): 5 samples with significant hemolysis, 5 samples with significant jaundice, and 5 samples with significant lipemia.

[0114] Methods: All samples were tested simultaneously using the respective test groups, control groups, and reference methods, and the relative deviations were calculated.

[0115] The experimental results are shown in Table 4.

[0116] Table 4: Performance Comparison of No and Added Anti-interference Buffer System

[0117] Analysis of the experimental data in Table 4 shows that 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. This demonstrates that the buffer system has a universal and crucial improving effect on homogeneous detection platforms.

[0118] Regarding the degree of performance degradation: Example 1 (HRP) deteriorated the most severely because it was most affected by endogenous substances.

[0119] Although Example 2 (ALP) showed improvement, the deviation was still unacceptable without an anti-interference system.

[0120] Example 3 (β-Gal) shows that its inherent advantages are severely weakened without an anti-interference system, with deviations in hemolysis and jaundice even exceeding those of the ALP system under system protection. This particularly demonstrates that the buffer system is not merely an added bonus, but a necessary condition for the β-Gal system to achieve its theoretically optimal performance.

[0121] Regarding performance recovery: After adding the anti-interference buffer system, the accuracy of all systems was greatly and decisively improved. Among them, Example 3 (β-Gal), combined with the anti-interference system, achieved near-perfect accuracy (average deviation <1%), highlighting the powerful synergistic effect of combining the "optimal enzyme system" and the "universal anti-interference technology".

[0122] In summary, Example 3 (β-Gal) is the optimal combination of the "anti-interference buffer system" and the homogeneous chemiluminescence detection system.

[0123] 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 I, 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 chemiluminescence kit also includes PGI calibrators and PGI quality control samples.

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

3. A chemiluminescent reagent kit for detecting pepsinogen I according to claim 2, characterized in that, The enzyme markers also include: HRP-labeled anti-PGI monoclonal antibody, bovine serum albumin, trehalose, sucrose, Proclin™ 300, and phosphate buffer.

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

5. A chemiluminescent reagent kit for detecting pepsinogen I according to claim 4, characterized in that, The enzyme markers also include: ALP-labeled anti-PGI monoclonal antibody, bovine serum albumin, trehalose, sucrose, magnesium chloride, Proclin™ 300, and Tris-HCl buffer.

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

7. A chemiluminescent reagent kit for detecting pepsinogen I according to claim 6, characterized in that, The enzyme markers also include: β-galactosidase-labeled anti-PGI monoclonal antibody, bovine serum albumin, trehalose, sucrose, magnesium chloride, sodium chloride, Proclin™ 300, and phosphate buffer.

8. A chemiluminescent reagent kit for detecting pepsinogen I 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.

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

10. A method for preparing a chemiluminescent reagent kit for detecting pepsinogen I, characterized in that, The preparation method of the reagent kit is as follows: S1: Preparation of enzyme-labeled antibody: 1.0 g / L of β-galactosidase-labeled anti-PGI monoclonal antibody, 50.0 g / L of trehalose, 20.0 g / L of sucrose, 10.0 g / L of bovine serum albumin, 0.5 g / L of magnesium chloride and 1.0 mL / L of 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 to remove bacteria, aliquoted and stored. 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-PGI 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 0.1-0.3 mM Galacton-Star chemiluminescent substrate, 20.0 g / L tris(hydroxymethyl)aminomethane, 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 2.0 mL / L Tween-20, 1.5 g / L mouse IgG, 0.05 g / L L-ascorbic acid, 500 U / L catalase 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 I antigen to a series of concentrations of 1.56-200 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.

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