CN-122017231-A - Chemiluminescent immunoassay kit for quantitatively detecting tPAI.C and detection method thereof

Abstract

The invention relates to the technical field of immunodetection, and discloses a chemiluminescent immunoassay kit for quantitatively detecting tPAI.C and a detection method thereof. The kit comprises streptavidin-coated magnetic beads, biotin-labeled and acridinium ester-labeled tPAI.C paired antibodies, a calibrator and a quality control product. The invention solves the problems of insufficient antibody specificity, easy influence by interferents and the like possibly existing in the existing chemiluminescence method when tPAI.C is detected by screening the paired antibodies with high affinity and high specificity and optimizing a reaction system. 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 immunoassay analyzer, and can provide 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.

Inventors

• YANG MENGXIA
• HUANG KAI
• ZHENG SHUJIAN

Assignees

• 杭州隆基生物技术有限公司

Dates

Publication Date

20260512

Application Date

20260226

Claims (10)

1. 1. A chemiluminescent immunoassay kit for quantitatively detecting tPAI.C is characterized by comprising 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 product.
2. 2. The chemiluminescent immunoassay kit for quantitatively detecting tPAI.C according to claim 1 wherein the streptavidin-coated magnetic bead working solution comprises streptavidin magnetic beads and a magnetic bead diluent, wherein the particle size of the streptavidin magnetic beads is 1.0-3.0 μm, the active group is amino or carboxyl, the working concentration of the streptavidin magnetic beads is 0.5-3 mg/mL, and the magnetic bead diluent comprises Tris-HCl, naCl, EDTA-2Na, casein Na, tween-20, bovine IgG, trehalose and Proclin 300, and the pH is 7.40+ -0.2.
3. 3. The chemiluminescent immunoassay kit of claim 1 wherein the working solution of biotin-labeled tPAI.C antibody comprises biotin-labeled tPAI.C antibody and biotin diluent, wherein the working concentration of the antibodies is 0.1-2 μg/mL, and the biotin diluent comprises Na 2 HPO 4 ·12H 2 O、NaH 2 PO 4 ·2H 2 O, naCl, EDTA-2Na, BSA, fish skin gelatin, tween-20 and Proclin 300, and the pH is 7.40+ -0.2.
4. 4. The chemiluminescent immunoassay kit of claim 1 wherein the acridine ester-labeled tPAI.C antibody working solution comprises an acridine ester-labeled tPAI.C antibody and an acridine ester diluent, wherein the working concentration of the antibody is 0.1-2 μg/mL, the acridine ester diluent comprises Na 2 HPO 4 ·12H 2 O、NaH 2 PO 4 ·2H 2 O、NaCl、EDTA-2Na、F68、Bovine IgG、Casein Na、BSA、Tween-20 and Proclin 300, and the pH is 6.20+ -0.2.
5. 5. The chemiluminescent immunoassay kit of claim 1 wherein the tPAI.C calibrator comprises 2 levels of 10ng/mL and 50ng/mL.
6. 6. The chemiluminescent immunoassay kit of claim 1 wherein the tPAI.C controller comprises 2 levels of target values ranging from 10.+ -. 2.5ng/mL to 50.+ -. 12.5ng/mL, respectively.
7. 7. A chemiluminescent immunoassay kit for quantitative detection of tpai.c according to any one of claims 1-6 wherein the preparation method comprises the steps of: (1) The preparation of streptavidin-coated magnetic bead working solution comprises the steps of diluting streptavidin magnetic beads to 0.5-3 mg/mL by using a magnetic bead diluent, stirring and uniformly mixing, and preserving; (2) The preparation of the working solution of the biotin-labeled tPAI.C antibody comprises the steps of dialyzing, biotin-labeling, purifying and diluting the tPAI.C antibody to obtain the working solution with the working concentration of 0.1-2 mug/mL; (3) The preparation of the working solution of the tPAI.C antibody marked by the acridine ester comprises the steps of diluting the tPAI.C antibody, marking by the acridine ester, stopping the reaction, purifying and diluting to obtain the working solution with the working concentration of 0.1-2 mug/mL; (4) And preparing a calibrator and a quality control product, namely diluting the tPAI.C standard product to a set concentration by using a calibrator diluent, and sub-packaging and storing.
8. 8. The method of claim 7, wherein the Biotin is labeled with Sulfo-NHS-LC-Biotin at a molar ratio of Biotin to antibody of 15:1.
9. 9. The method of claim 7, wherein the acridinium ester is NHS ester, and the molar ratio of the acridinium ester to the antibody is 10:1.
10. 10. A method of detecting tpai.c using the kit of any one of claims 1-6, comprising the steps of: (1) The preparation of the instrument comprises the steps of starting a full-automatic chemiluminescence immunoassay analyzer, and loading consumable materials and reagents; (2) Adding 10 mu L of test sample, 100 mu L of biotin marker working solution and 100 mu L of acridinium ester marker working solution into a reaction tube, incubating for 10min at 37+/-0.3 ℃, and then adding 50 mu L of magnetic bead working solution, and incubating for 5min at 37+/-0.3 ℃; (3) Magnetic separation cleaning, namely placing a reaction tube on a magnetic separation frame for standing for 1min, removing supernatant, sequentially adding 200 mu L of cleaning solution twice, standing for 1min each time, removing supernatant, adding 200 mu L of cleaning solution for the third time, transferring the solution to a luminous tube, and standing for 1min, and removing supernatant; (4) Luminescence detection, namely adding chemiluminescent excitation liquid to detect the luminous intensity, and automatically calculating the concentration of tPAI.C by an instrument through a calibration curve.

Description

Chemiluminescent immunoassay kit for quantitatively detecting tPAI.C and detection method thereof Technical Field The invention belongs to the technical field of immunodetection analysis, and particularly relates to a chemiluminescent immunoassay kit for quantitatively detecting tPAI.C and a detection method thereof. Background The tissue type plasminogen activator-plasminogen activator inhibitor-1 complex (tissue plasminogen activator-plasminogen activator inhibitor-1 complex, tpai.c) is a stable protein complex formed by covalent binding of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the plasmatic system when vascular endothelial cells are damaged. The compound is the product of endothelial injury and activation of fibrinolytic system, and is also a core molecular marker for reflecting the activation of vascular endothelial injury and fibrinolytic system, and the level change of the compound can indirectly reflect the dynamic state of body coagulation and fibrinolytic system. T-PA is a single chain glycoprotein, which has a certain regulation effect in fibrinolytic system. the t-PA is mainly synthesized and secreted by vascular endothelial cells and becomes a plasminogen activator after entering the blood of a human body, so that the plasminogen can be converted into plasmin, and the coagulation factors and the fibrin are degraded, thereby ensuring the smoothness of the blood vessel. In the occurrence and development of thrombotic diseases, t-PA plays a certain role in inhibition, so that in a normal organism, t-PA is in a high expression state. PAI-1 is the most important inhibitor of plasmatic fibrinolytic activity, and can inhibit free t-PA, and its level increase promotes fibrin deposition and thrombosis. In the process of regulating and maintaining the balance of a fibrinolytic system of a body, PAI-1 and t-PA are two important factors, and the body has certain balance between the t-PA and the PAI-1 level in circulating blood under normal state, and once the t-PA is released into human blood, the t-PA is combined by the PAI-1 for the first time, so that the integrity of blood vessels and the smoothness of the blood vessels are protected. However, imbalance between t-PA and PAI-1 levels can occur as a series of pathological changes. The prognosis of the disease can be judged clinically by tpai.c. the elevation of tpai.c levels is closely related to many cardiovascular diseases, such as atherosclerosis, coronary heart disease, primary hypertension, deep vein thrombosis, pulmonary thromboembolism, cerebral thrombosis, septic shock, etc., and is a sensitive index for evaluating the efficacy of anticoagulation or thrombolysis treatment, screening and diagnosing hemorrhagic diseases, providing a key basis for clinical decision. The existing detection methods of tPAI.C in clinic include a latex enhanced immunoturbidimetry, a radioimmunoassay, an enzyme-linked immunosorbent assay, a chemiluminescent method and the like. The emulsion enhanced immunoturbidimetry has the advantages of simple operation, high safety, but poor sensitivity, and is difficult to detect samples with extremely low concentration. The radioimmunoassay has high sensitivity and high specificity, but is complex to operate, high in cost and easy to cause radioactive pollution. Although the ELISA method has better reagent specificity and high safety, the operation steps are complicated, the time consumption is long, the batch detection efficiency is low, and the sensitivity is lower than that of the radioimmunoassay. The chemiluminescence method has the advantages of high sensitivity, high detection speed, high automation degree and high safety, but the existing chemiluminescence kit still has the problems of insufficient antibody affinity, insufficient specificity, easiness in being influenced by interferents and the like. Therefore, the kit for quantitatively detecting the tissue type plasminogen activator-plasminogen activator inhibitor-1 complex is provided with higher performance and stability by innovatively screening the high-affinity tPAI.C antibody pair, optimizing the labeling process and improving the buffer system, so as to meet the clinical requirement for accurate detection of the tPAI.C. Disclosure of Invention The invention provides a chemiluminescent immunoassay kit for quantitatively detecting tPAI.C and a detection method thereof, and aims to solve the problems. In order to solve the problems, the chemiluminescent immunoassay kit for quantitatively detecting the tPAI.C and the detection method thereof provided by the invention adopt a double-antibody sandwich magnetic particle chemiluminescent immunoassay method, and the kit comprises streptavidin-coated magnetic bead working solution, biotin-labeled tPAI.C antibody working solution, acridine ester-labeled tPAI.C antibody working solution, tPAI.C calibrator and tPAI.C quality control product. Preferably, in the streptavidin-coat