Chemiluminescence immunoassay kit for quantitative detection of tPAI.C and detection method thereof

Abstract

The invention relates to the technical field of immunodetection, and discloses a chemiluminescence immunoassay kit for quantitatively detecting tPAI.C and a detection method thereof. The kit comprises magnetic beads coated with streptavidin, a tPAI.C pairing antibody labeled by biotin and acridinium ester, a calibration product and a quality control product. By screening high-affinity and high-specificity paired antibodies and optimizing a reaction system, the problems that when tPAI.C is detected by an existing chemiluminescence method, the antibodies are insufficient in specificity, prone to being influenced by interferents and the like are solved. The kit has the characteristics of high sensitivity, strong specificity, good accuracy, excellent anti-interference capability and good stability, is suitable for a full-automatic chemiluminescence immunity analyzer, and can provide a reliable basis for clinical auxiliary diagnosis, curative effect monitoring and prognosis evaluation of diseases such as acute thrombotic diseases, disseminated intravascular coagulation (DIC), severe sepsis and the like.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay analysis technology, and particularly relates to a chemiluminescent immunoassay kit for quantitative detection of tPAI.C and its detection method. Background Technology

[0002] The tissue plasminogen activator-plasminogen activator inhibitor-1 complex (tPAI.C) is a stable protein complex formed by the covalent binding of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the fibrinolytic system during vascular endothelial cell injury. This complex is a product of endothelial injury and fibrinolytic system activation, and is also a core molecular marker reflecting vascular endothelial injury and fibrinolytic system activation; changes in its level can indirectly reflect the dynamic state of the body's coagulation and fibrinolytic systems.

[0003] t-PA is a single-chain glycoprotein that plays a regulatory role in the fibrinolytic system. t-PA is mainly synthesized and secreted by vascular endothelial cells. After entering the bloodstream, it becomes a plasminogen activator, converting plasminogen into plasmin, which degrades coagulation factors and fibrin, thus ensuring vascular patency. t-PA plays an inhibitory role in the development of thrombotic diseases; therefore, it is highly expressed in normal individuals. PAI-1 is the most important inhibitor of plasma fibrinolytic activity, inhibiting free t-PA. Increased t-PA levels promote fibrin deposition and thrombus formation. PAI-1 and t-PA are two important factors in regulating and maintaining the balance of the fibrinolytic system. Under normal conditions, there is a balance between t-PA and PAI-1 levels in the circulating blood. Once t-PA is released into the bloodstream, it is immediately bound to PAI-1, thus protecting the integrity and patency of blood vessels. However, an imbalance between t-PA and PAI-1 levels can lead to a series of pathological changes.

[0004] Clinically, tPAI.C can be used to assess the prognosis of diseases. Elevated tPAI.C levels are closely associated with many cardiovascular diseases, such as atherosclerosis, coronary heart disease, essential hypertension, deep vein thrombosis, pulmonary embolism, cerebral thrombosis, and septic shock. It is a sensitive indicator for evaluating the efficacy of anticoagulation or thrombolytic therapy, screening for and diagnosing hemorrhagic diseases, and provides crucial information for clinical decision-making.

[0005] Currently available clinical methods for detecting tPAI.C include latex-enhanced immunoturbidimetry, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay. While latex-enhanced immunoturbidimetry is simple to operate and highly safe, it suffers from poor sensitivity and difficulty detecting extremely low concentrations. Radioimmunoassay offers high sensitivity and specificity, but is complex, costly, and prone to radioactive contamination. ELISA, while offering good reagent specificity and safety, is cumbersome, time-consuming, has low batch processing efficiency, and lower sensitivity than radioimmunoassay. Chemiluminescence immunoassay offers high sensitivity, fast detection speed, high automation, and high safety, but existing chemiluminescence kits still suffer from insufficient antibody affinity, insufficient specificity, and susceptibility to interfering substances. Therefore, this invention innovatively screens high-affinity tPAI.C antibody pairs, optimizes the labeling process, and improves the buffer system to provide a higher-performance, more stable kit for the quantitative detection of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, meeting the clinical need for accurate tPAI.C detection. Summary of the Invention

[0006] This invention provides a chemiluminescent immunoassay kit and its detection method for quantitative detection of tPAI.C, aiming to solve the above-mentioned problems.

[0007] To address the aforementioned problems, this invention provides a chemiluminescent immunoassay kit and method for the quantitative detection of tPAI.C, employing a double-antibody sandwich magnetic microparticle chemiluminescent immunoassay. The kit comprises: streptavidin-coated magnetic bead working solution, biotin-labeled tPAI.C antibody working solution, acridinium ester-labeled tPAI.C antibody working solution, tPAI.C calibrator, and tPAI.C quality control sample.

[0008] Preferably, in the working solution of the streptavidin-coated magnetic beads, 20 μg / mg streptavidin magnetic beads (particle size 1.0-3.0 μm, active group is amino or carboxyl, working concentration 0.5-3 mg / mL) are suspended in a magnetic bead diluent, wherein the magnetic bead diluent includes Tris-HCl, NaCl, EDTA-2Na, Casein Na, Tween-20, Bovine IgG, trehalose and Proclin 300, and the pH is 7.40±0.2.

[0009] Preferably, the working solution of the biotin-labeled tPAI.C antibody consists of biotin-labeled tPAI.C antibody + biotin diluent, with an antibody working concentration of 0.1-2 μg / mL. The biotin diluent includes Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, EDTA-2Na, BSA, fish skin gelatin, Tween-20, and Proclin 300, with a pH of 7.40±0.2.

[0010] Biotin dilution solution:

[0011] The above materials were added to purified water in proportion, stirred thoroughly to dissolve, and the pH was adjusted to 7.40±0.2.

[0012] Preferably, the working solution of the acridine ester-labeled tPAI.C antibody consists of acridine ester-labeled tPAI.C antibody + acridine ester diluent, with an antibody working concentration of 0.1~2 μg / mL. The acridine ester diluent includes Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, EDTA-2Na, F68, Bovine IgG, Casein Na, BSA, Tween-20, and Proclin 300, with a pH of 6.20±0.2.

[0013] acridine ester diluent:

[0014] The above materials were added to purified water in proportion, stirred thoroughly to dissolve, and the pH was adjusted to 6.20±0.2.

[0015] tPAI.C calibrators: 2 levels, 10 ng / mL and 50 ng / mL, with a phosphate buffer containing 0.5% BSA and a pH of 7.40 ± 0.1.

[0016] tPAI.C quality control: 2 levels, 10±2.5 ng / mL and 50±12.5 ng / mL, with a matrix of phosphate buffer containing 0.5% BSA and a pH of 7.40±0.1.

[0017] Preferably, the preparation method of the magnetic bead working solution includes the following steps: diluting streptavidin magnetic beads with magnetic bead diluent to 0.5~3 mg / mL, stirring and mixing, and then storing. The magnetic bead diluent includes Tris-HCl, NaCl, EDTA-2Na, Casein Na, Tween-20, Bovine IgG, trehalose, and Proclin 300, with a pH of 7.40±0.2.

[0018] The magnetic bead diluent is prepared as follows:

[0019] The above materials were added to purified water in proportion, stirred thoroughly to dissolve, and the pH was adjusted to 7.40±0.2.

[0020] Preferably, the preparation method of the biotin-labeled tPAI.C antibody working solution includes the following steps: Dialyze the tPAI.C antibody with 1× biotinylated buffer; Then, Sulfo-NHS-LC-Biotin was added at a biotin:antibody molar ratio of 15:1 to react with it, and then purified by dialysis to obtain the biotin-labeled product, which was then stored in 50% glycerol (volume fraction). The biotin-labeled compound was mixed with the biotin dilution solution and stored to obtain the working solution of the biotin-labeled compound.

[0021] The 1× biotin-labeled buffer was obtained by diluting a 10× biotin-labeled buffer, wherein the 10× biotin-labeled buffer comprises NaH2PO4·2H2O, Na2HPO4·12H2O, and NaCl, with a pH of 8.50±0.2; the biotin diluent comprises Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, EDTA-2Na, BSA, fish skin gelatin, Tween-20, and Proclin 300, with a pH of 7.40±0.2.

[0022] Preferably, the preparation method of the acrid ester-labeled tPAI.C antibody working solution includes the following steps: Dilute the tPAI.C antibody with acridine ester labeling buffer; Acridinium ester and DMF were added at a molar ratio of acridinium ester to antibody of 10:1, and the reaction was terminated with lysine. The acridine ester label was purified by dialyzing with 1× acridine ester dialysis buffer and then stored in acridine ester antibody preservation solution and 50% glycerol (volume fraction). The biotin-labeled compound was mixed with acridine ester dilution solution and stored to obtain the working solution of acridine ester labeling.

[0023] The acridinium ester labeling buffer comprises NaHCO3 and EDTA-2Na, with a pH of 9.50±0.1; the 1× acridinium ester dialysis buffer is obtained by diluting 10× acridinium ester dialysis buffer, which comprises NaH2PO4·2H2O, Na2HPO4·12H2O, NaCl, and EDTA-2Na, with a pH of 7.40±0.1; the acridinium ester antibody preservation solution comprises Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, BSA, and Proclin 300, with a pH of 6.20±0.2; the acridinium ester dilution solution comprises Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, EDTA-2Na, F68, Bovine IgG, Casein Na, BSA, Tween-20, and Proclin 300. 300, pH 6.20±0.2.

[0024] acridinium ester antibody preservation solution

[0025] The above materials were added to purified water in proportion, stirred thoroughly to dissolve, and the pH was adjusted to 6.20±0.2.

[0026] Preferably, the preparation method of the tPAI.C calibrator includes the following steps: 1) Dilute the tPAI.C antigen with calibrator diluent to prepare the tPAI.C calibrator intermediate solution; 2) Dilute the tPAI.C calibrator intermediate solution with calibrator diluent to 10 ng / mL (standard 1) and 50 ng / mL (standard 2); The calibrator diluent comprises 0.5% bovine serum albumin (BSA) in phosphate buffer, pH=7.40±0.2.

[0027] Preferably, the preparation method of the tPAI.C quality control product is as follows: the tPAI.C calibrator intermediate solution is diluted with calibrator diluent to 10±2.5 ng / mL (quality control product L) and 50±12.5 ng / mL (quality control product M).

[0028] The present invention also provides a method for preparing the above-mentioned reagent kit, comprising the following steps: Preparation of working solution for streptavidin-coated magnetic beads; Preparation of working solution for biotin-labeled tPAI.C antibody; Preparation of acridine ester-labeled tPAI.C antibody working solution; Prepare tPAI.C calibrators and quality control samples.

[0029] This invention also provides a method for detecting tissue plasminogen activator-plasminogen activator inhibitor-1 complex (tPAI.C) using the above-described kit, comprising the following steps: (1) Instrument preparation: Start the fully automated chemiluminescence immunoassay analyzer and load consumables and reagents; (2) Sample addition and incubation: Add 10 μL of test sample, 100 μL of biotin-labeled working solution and 100 μL of acridinium ester-labeled working solution to the reaction tube, and incubate at 37℃ (±0.3°C) for 10 min; then add 50 μL of magnetic bead working solution, and continue incubating at 37℃ (±0.3°C) for 5 min; (3) Magnetic separation cleaning: Place the reaction tube on the magnetic separation rack and let it stand for 1 min to remove the supernatant; add 200 μL of cleaning solution twice in sequence, and let it stand for 1 min each time before removing the supernatant; add 200 μL of cleaning solution for the third time, transfer the solution to the light-emitting tube, let it stand for 1 min and then remove the supernatant. (4) Luminescence detection: Add chemiluminescence excitation solution, detect luminescence intensity, and the instrument automatically calculates the tPAI.C concentration through the calibration curve.

[0030] The present invention has the following beneficial technical effects: Excellent detection performance with high sensitivity and specificity: By screening for high-affinity specific antibody pairs and optimizing the labeling process, the kit ensures excellent sensitivity and specificity. Its limits of detection (LoD) are all no higher than 1.0 ng / mL, enabling effective detection of low-concentration samples and sensitive monitoring of early or mild vascular endothelial cell damage, providing possibilities for early disease diagnosis. Employing a double-antibody sandwich method and selecting monoclonal antibodies targeting different epitopes of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex, cross-reaction with non-target substances is minimized, ensuring high specificity of the detection results.

[0031] Strong resistance to interference: It can tolerate 20 mg / dL bilirubin (jaundice), 2000 mg / dL triglycerides (lipemia), 500 mg / dL hemoglobin (hemolysis), 1000 ng / mL HAMA and 1500 IU / mL rheumatoid factor (RF), avoiding false positives / false negatives caused by interference from clinical samples.

[0032] No Hook effect: No hook effect was observed in high-concentration tPAI.C samples (i.e., the detected value was not lower than 90% of the true value), making it suitable for detection in patients with severe vascular endothelial damage (such as severe sepsis).

[0033] High precision, good repeatability and consistency: This invention uses magnetic microparticles as a solid-phase carrier, which has a large surface area and uniform binding. Combined with an optimized reaction system, the kit has high precision. The intra-batch coefficient of variation (CV) is no greater than 8%, and the inter-batch coefficient of variation (CV) is no greater than 10%. This shows that the kit can maintain a high degree of consistency and stability of results at different times and by different operators, providing a guarantee for longitudinal comparison and long-term monitoring of clinical results.

[0034] With good stability, it is suitable for fully automated platforms: By optimizing the preservation solution formulation of the labeled antibodies (such as adding glycerol, BSA, Proclin 300, etc.), the activity of the labeled antibodies is effectively protected and the shelf life of the kit is extended. The kit components are designed to be fully compatible with mainstream fully automated chemiluminescence immunoassay analyzers on the market, realizing full automation of the entire process from sample addition, incubation, cleaning, detection to result calculation. This frees operators from tedious manual operations, greatly improves detection efficiency, and reduces human error, meeting the high-throughput and standardized needs of modern clinical testing.

[0035] In summary, this invention provides a chemiluminescent immunoassay kit and its detection method for the quantitative detection of tPAI.C. This kit exhibits high sensitivity, high specificity, high accuracy, strong anti-interference ability, and excellent stability. It can be used as an auxiliary diagnostic tool in clinical practice for diseases such as acute thrombotic diseases, severe sepsis, severe trauma, and disseminated intravascular coagulation (DIC), providing a reference for clinical intervention and prognosis. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the structure of a kit for quantitative detection of tPAI.C provided by the present invention; Figure 2 This is the linear fitting curve of the tPAI.C quantitative detection kit of the present invention. Detailed Implementation

[0037] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0038] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of those skilled in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or identical to those described in the embodiments of this invention may be used to implement this invention.

[0039] Unless otherwise stated, the test methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0041] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0042] Example 1 This invention provides a kit for the quantitative detection of tPAI.C, such as... Figure 1 As shown, its preparation method includes the following steps: 1. Preparation of streptavidin-coated magnetic bead working solution The magnetic microparticle carrier can also be a solid-phase carrier, a microplate, a plastic tube, or a plastic bead; this kit preferably uses magnetic microparticles: due to their large surface area and strong protein binding ability, the magnetic microparticles have a particle size of 1.0~3.0μm, the active group is amino or carboxyl, and the concentration of the magnetic microparticles used is 0.5~3mg / mL.

[0043] 1.1 Preparation of magnetic bead diluent: Tris-HCl 3.0285g, NaCl 8.766g, EDTA-2Na 0.3722g, Casein Na 5g, Tween-20 1mL, Bovine IgG 1g, Trehalose 1g, Proclin 300 0.5mL; add the above materials to 1000mL of purified water, stir thoroughly to dissolve, and adjust the pH to 7.40±0.2; 1.2 Preparation of magnetic bead working solution: Take 0.5~3mg of streptavidin magnetic beads, add 1mL of magnetic bead diluent, vortex and mix well to obtain the magnetic bead working solution.

[0044] 2. Preparation of biotin-labeled tPAI.C antibody working solution 2.1.1 Preparation of biotin-labeled buffer (10×): 0.74g NaH2PO4·2H2O, 34.11g Na2HPO4·12H2O, 87.66g NaCl; add the above materials to 1000mL purified water, stir thoroughly to dissolve, adjust the pH to 8.50±0.2, and dilute to 1× before use; 2.1.2. Preparation of biotinylated label: 1-5 mg of tPAI.C antibody (mouse monoclonal antibody, protein site: kringle 2 domain), appropriate amount of Sulfo-NHS-LC-Biotin (biotin:antibody molar ratio = 15:1), 4000 mL of 1×biotin-labeled buffer, and 50% glycerol (volume fraction). The preparation method is as follows:

[0045] 1) Dilute the antibody with 1× Biotinylated buffer to 1~5 mg / mL, add it to the dialysis bag and mix well. Dialyze in 1× Biotinylated buffer at room temperature for 2 hours, then change to 1× Biotinylated buffer and dialyze for another 2 hours. 2) Prepare 100 μL of 1~5 mg / mL Sulfo-NHS-LC-Biotin (biotin: antibody molar ratio = 15:1), add it to the dialysis bag, mix well, and react at room temperature for 1 hour; 3) Dialyze overnight with 1× biotinylated buffer; 4) Remove the biotin-labeled product from the dialysis bag and add it to an appropriate container. Then, add 1× biotin-labeled buffer to bring the volume up to a final concentration of 0.5~5 mg / mL. 5) Add 50% by volume of glycerol to the labeled product, mix well by blowing, and store in a refrigerator at 4°C for later use, or store in a refrigerator at -20°C for a long time.

[0046] 2.2 Preparation of Biotin Diluent: 3.58g Na2HPO4·12H2O, 0.468g NaH2PO4·2H2O, 8.766g NaCl, 0.3722g EDTA-2Na, 5g BSA, 10g fish skin gelatin, 1mL Tween-20, 0.5mL Proclin 300; add the above materials to 1000mL purified water, stir thoroughly to dissolve, and adjust the pH to 7.40±0.2; 2.3 Preparation of working solution for biotin labeling: Take an appropriate amount of biotin labeling, add 1 mL of biotin diluent, and mix well.

[0047] 3. Preparation of acridinium ester-labeled tPAI.C antibody working solution 3.1.1 Preparation of acridinium ester labeling buffer: 1.6802 g NaHCO3, 0.3722 g EDTA-2Na; add the above materials to 1000 mL purified water, stir thoroughly to dissolve, and adjust the pH to 9.50 ± 0.1; 3.1.2 Preparation of acridinium ester dialysis buffer (10×): 4.1g NaH2PO4·2H2O, 28.39g Na2HPO4·12H2O, 87.66g NaCl, and 1.8612g EDTA-2Na were added to 1000mL of purified water and stirred thoroughly to dissolve. The pH was adjusted to 7.40±0.1. The solution was diluted to 1× before use. 3.1.3 Preparation of acridinium ester antibody preservation solution: 3.5814 g Na2HPO4·12H2O, 0.39 g NaH2PO4·2H2O, 8.766 g NaCl, 5 g BSA, and 0.5 mL Proclin 300 were added to 1000 mL of purified water, stirred thoroughly to dissolve, and the pH was adjusted to 6.20 ± 0.1. 3.1.4. Acridinium ester labeling compound: 1-5 mg tPAI.C antibody, appropriate amount of acridinium ester (NHS ester), appropriate amount of acridinium ester labeling buffer, 10 L 1× acridinium ester dialysis buffer, appropriate amount of acridinium ester antibody preservation solution. The preparation method of the acridinium ester labeling compound is as follows: 1) Place the tPAI.C antibody (mouse monoclonal antibody, protein site: reaction center loop RCL) in a centrifuge tube, dilute it to 1~5 mg / mL with acridinium ester labeling buffer, add acridinium ester and DMF at a molar ratio of 10:1 (acriminium ester: antibody), and mix at 70 r / min in the dark for 2 h. 2) After the reaction is complete, add 1% volume of 1M lysine to the solution and mix at 70 r / min in the dark for 30 min. 3) Pour 1L of 1× acridine ester dialysis buffer into a 1L beaker, add the acridine ester labeled product from 2) into the dialysis bag, mix gently, and place in the acridine ester dialysis buffer for dialysis at room temperature for 2 hours. The dialysis process should be carried out in a dark environment. 4) Change the acridinium ester dialysate every 2 hours, repeat 10 times; 5) After dialysis, remove the acridinium ester labeled product from the dialysis bag, place it in a centrifuge tube, add 10% volume of acridinium ester antibody preservation solution and 50% volume of glycerol, mix well by pipetting, and store in a 4°C refrigerator for later use, or store in a -20°C refrigerator for long-term storage.

[0048] 3.2 Preparation of acridinium ester diluent: Na₂HPO₄·12H₂O 3.5814g, NaH₂PO₄·2H₂O 0.468g, NaCl 8.766g, EDTA-2Na 0.3722g, F68 1g, Bovine IgG 1g, Casein Na 5g, BSA 5g, Tween-20 1mL, Proclin 300 0.5mL; add the above materials to 1000mL of purified water, stir thoroughly to dissolve, and adjust the pH to 6.20±0.2; 3.3. Acridine ester labeling working solution: Take an appropriate amount of acridine ester labeling, add 1 mL of acridine ester diluent, and mix well.

[0049] 4. Preparation of tPAI.C calibrators 4.1 Calibration Diluent The diluent is a phosphate buffer containing 0.5% bovine serum albumin (BSA) with a pH of 7.40 ± 0.1.

[0050] 4.2 tPAI.C calibrators 1) Dilute the tPAI.C calibrator with calibrator diluent to prepare the tPAI.C calibrator intermediate solution; 2) Dilute the tPAI.C calibrator intermediate solution with calibrator diluent to 10 ng / mL (standard 1) and 50 ng / mL (standard 2); 5. Preparation of tPAI.C quality control samples The tPAI.C calibrator intermediate solution was diluted with calibrator diluent to (10±2.5) ng / mL (quality control L) and (50±12.5) ng / mL (quality control M).

[0051] Preparation of cleaning solution: Tris-HCl buffer (pH 7.4) containing 0.05%~0.1% Tween-20; Preparation of chemiluminescence excitation solution: including substrate solution A (containing 0.05~0.2M nitric acid and 0.3~0.5% hydrogen peroxide) and substrate solution B (containing 0.2~0.3M NaOH and 0.6~1% cetrimonium chloride).

[0052] Example 2 Reagent kit performance testing Test methods Instrument: Fully automated chemiluminescence immunoassay analyzer Before starting the test, check that the instrument has successfully completed its self-test and prepare the necessary materials. The basic test procedures are as follows: 1) Loading consumables, including consumable boxes, reaction cup boxes, substrate bottles, and reagent boats; 2) Load at least 200 μL of sample. Enter the sample request interface and input the relevant information; 3) Tap [Start], select the test sample and test item, and enter the number of repetitions; 4) The instrument reads the sample information and test items, and then automatically performs the sample test; 5) The instrument will aspirate 10 μL of the sample to be tested, 100 μL of biotin-labeled working solution, and 100 μL of acridinium ester-labeled working solution into a reaction vessel, incubate at 37°C (±0.3°C) for 10 minutes, then wash. Add 50 μL of magnetic bead working solution, incubate at 37°C (±0.3°C) for 5 minutes, then wash. Add substrate solutions A and B for the fully automated immunoassay system, and measure the luminescence signal.

[0053]

[0054] Enter the results interface to view the test results.

[0055] 1. Accuracy: The tPAI.C standard was prepared into two concentrations, 10 ng / mL and 50 ng / mL, using phosphate buffer containing 0.5% bovine serum albumin. Each concentration sample was measured three times. The test result was recorded as (Xi), calculated using the formula: Bi = (Xi – T) / T × 100% Calculate the relative deviation (Bi) separately. The relative deviation between the measured concentration and the labeled value should be within ±10%. The test results are shown in the table below: Table 1: Accuracy Results of the Tissue Plasminogen Activator-Plasminogen Activator Inhibitor-1 Complex Reagent

[0056] 2. Limit of detection: Not higher than 1.0 ng / mL; 3. Linearity: The linear correlation coefficient (r) is not less than 0.9900 in the range of 1~100 ng / mL.

[0057] Take one high-value sample of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex that is close to the upper limit of the linear interval, and dilute it into 5 concentration samples according to a certain ratio. The low-value concentration sample is close to the lower limit of the linear interval. Repeat the above samples twice, calculate the average value, and fit the average value of the measured concentration to the theoretical concentration using the least squares method. Calculate the linear correlation coefficient r. If the correlation coefficient r is not less than 0.9900, the linear setting is considered reasonable.

[0058] Table 2: Linearity Validation Results of the Tissue Plasminogen Activator-Plasminogen Activator Inhibitor-1 Complex Reagent

[0059] Linear relationship such as Figure 2 As shown.

[0060] 4. Repeatability: Coefficient of variation (CV) not greater than 8%; 5. Inter-batch variation: The inter-batch coefficient of variation (CV) is no greater than 10%; 6. Interfering Substances: Interfering substances were added to clinical samples to prepare appropriate concentrations. The control sample without interfering substances and the sample with interfering substances were measured three times, and the average value and deviation were calculated. Deviation was calculated as follows: Deviation = (Detection concentration after adding interfering substance - Control group concentration) / Control group concentration x 100%. The deviation should be within ±10%. The test results were not affected by jaundice (20 mg / dL bilirubin), lipemia (2000 mg / dL triglycerides), hemolysis (500 mg / dL hemoglobin), human anti-mouse antibody (HAMA) 1000 ng / mL, or rheumatoid factor (RF 1500 IU / mL).

[0061] Table 3: Results of Reagent Interference Validation for the Tissue Plasminogen Activator-Plasminogen Activator Inhibitor-1 Complex

[0062] 7. Hook effect: No hook effect was observed at a concentration of 1600 ng / mL.

[0063] Specificity: Samples of tissue plasminogen activator (t-PA) at a concentration of 6 ng / mL, plasminogen activator inhibitor-1 (PAI-1) at a concentration of 40 ng / mL, and urokinase-type plasminogen activator-plasminogen activator-inhibitor-1 complex (uPA-PAI-1 complex) at a concentration of 50 ng / mL were measured once each, and the result should not exceed 1 ng / mL.

[0064] Table 4: Results of reagent specificity verification of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex

[0065] In Table 4, '<1' indicates that the detection value is lower than the detection limit of this kit (1.0 ng / mL), meaning it was not detected.

[0066] The stability of the kit in this embodiment was tested by placing it at 37°C for 7 days. Low, medium and high value samples were measured three times at 0 days, 3 days, 5 days and 7 days respectively. The average value was taken and compared with the test results of fresh comparative reagent.

[0067] Table 5: Results of reagent stability verification of the tissue plasminogen activator-plasminogen activator inhibitor-1 complex

[0068] The results show that the degradation rate of the reagents in the examples is less than that of the reagents in the comparative examples, proving that the stability of the reagents of the present invention can meet the requirements of the product.

[0069] The results above show that the performance test results of the kit involved in this invention are very close to those of similar kits, achieving excellent results. The tPAI.C chemiluminescent immunoassay kit of this invention has good applicability and advanced features.

[0070] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0071] It should be understood that the disclosed apparatus can be implemented in other ways, as illustrated in the embodiments provided in this application. For example, the apparatus embodiments described above are merely illustrative; the division of units described above is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or communication connections shown or discussed may be through some interfaces; the indirect coupling or communication connections between devices or units may be telecommunications or other forms.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A chemiluminescent immunoassay kit for quantitative detection of tPAI.C, characterized in that: This includes streptavidin-coated magnetic bead working solution, biotin-labeled tPAI.C antibody working solution, acridine ester-labeled tPAI.C antibody working solution, tPAI.C calibrators, and tPAI.C quality control products.

2. The chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in claim 1, characterized in that: The working solution of the streptavidin-coated magnetic beads consists of streptavidin magnetic beads and a magnetic bead diluent. The streptavidin magnetic beads have a particle size of 1.0~3.0μm and an active group of amino or carboxyl. The working concentration of the streptavidin magnetic beads is 0.5~3mg / mL. The magnetic bead diluent includes Tris-HCl, NaCl, EDTA-2Na, Casein Na, Tween-20, Bovine IgG, trehalose, and Proclin 300, with a pH of 7.40±0.

2.

3. The chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in claim 1, characterized in that: The biotin-labeled tPAI.C antibody working solution consists of biotin-labeled tPAI.C antibody and biotin diluent, with an antibody working concentration of 0.1~2 μg / mL; the biotin diluent includes Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, EDTA-2Na, BSA, fish skin gelatin, Tween-20 and Proclin 300, with a pH of 7.40±0.

2.

4. The chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in claim 1, characterized in that: The acridine ester-labeled tPAI.C antibody working solution consists of acridine ester-labeled tPAI.C antibody and acridine ester diluent, with an antibody working concentration of 0.1~2 μg / mL; the acridine ester diluent includes Na2HPO4·12H2O, NaH2PO4·2H2O, NaCl, EDTA-2Na, F68, Bovine IgG, Casein Na, BSA, Tween-20 and Proclin 300, with a pH of 6.20±0.

2.

5. The chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in claim 1, characterized in that: The tPAI.C calibrator includes two levels: 10 ng / mL and 50 ng / mL.

6. The chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in claim 1, characterized in that: The tPAI.C quality control material includes two levels: the target values ​​are 10±2.5 ng / mL and 50±12.5 ng / mL, respectively.

7. The chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in any one of claims 1-6, characterized in that, The preparation method includes the following steps: (1) Preparation of working solution for streptavidin-coated magnetic beads: dilute streptavidin magnetic beads with magnetic bead diluent to 0.5~3mg / mL, stir and mix well and store; (2) Preparation of working solution of biotin-labeled tPAI.C antibody: After dialysis, biotin labeling, purification and dilution, tPAI.C antibody was used to obtain working solution with a working concentration of 0.1~2μg / mL; (3) Preparation of working solution of acridine ester labeled tPAI.C antibody: after dilution, acridine ester labeling, termination of reaction, purification and dilution, working solution with working concentration of 0.1~2μg / mL was obtained; (4) Preparation of calibrators and quality control products: Dilute the tPAI.C standard to the set concentration with calibrator diluent and store it in aliquots.

8. The method for preparing the chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in claim 7, characterized in that: The biotin labeling used was Sulfo-NHS-LC-Biotin, with a biotin to antibody molar ratio of 15:

1.

9. The method for preparing the chemiluminescent immunoassay kit for quantitative detection of tPAI.C as described in claim 7, characterized in that: The acridine ester labeling uses acridine ester NHS ester, and the molar ratio of acridine ester to antibody is 10:

1.

10. A method for detecting tPAI.C using the kit described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Instrument preparation: Start the fully automated chemiluminescence immunoassay analyzer and load consumables and reagents; (2) Sample addition and incubation: Add 10 μL of test sample, 100 μL of biotin-labeled working solution and 100 μL of acridinium ester-labeled working solution to the reaction tube, and incubate at 37±0.3°C for 10 min; then add 50 μL of magnetic bead working solution and continue incubating at 37±0.3°C for 5 min. (3) Magnetic separation cleaning: Place the reaction tube on the magnetic separation rack and let it stand for 1 min to remove the supernatant; add 200 μL of cleaning solution twice in sequence, and let it stand for 1 min each time before removing the supernatant; add 200 μL of cleaning solution for the third time, transfer the solution to the light-emitting tube, let it stand for 1 min and then remove the supernatant. (4) Luminescence detection: Add chemiluminescence excitation solution, detect luminescence intensity, and the instrument automatically calculates the tPAI.C concentration through the calibration curve.

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