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An enzyme-linked immunoassay kit for human phosphorylated vasodilator-stimulating protein and its detection method

An enzyme-linked immunosorbent assay and vasodilation technology, applied in the field of molecular immunology, can solve the problems of complex process, high preparation cost, unfavorable large-scale industrial production, etc., and achieve the effects of simple detection steps, high sensitivity, and stable and repeatable results.

Active Publication Date: 2022-04-01
湖南菲思特精准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pre-preparation costs of these published detection methods are relatively high, and the process is relatively complicated, which is not conducive to large-scale industrial production.

Method used

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  • An enzyme-linked immunoassay kit for human phosphorylated vasodilator-stimulating protein and its detection method
  • An enzyme-linked immunoassay kit for human phosphorylated vasodilator-stimulating protein and its detection method
  • An enzyme-linked immunoassay kit for human phosphorylated vasodilator-stimulating protein and its detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the preparation of kit

[0039] The kit of the invention adopts the ELISA method and cooperates with a microplate reader, and is used for measuring the content of human phosphorylated blood vessel dilation-stimulated phosphoprotein in human body samples. The technical principle of the reaction is as follows: the VASP-P level in the serum is determined by the double-antibody sandwich method for the sample to be tested. Coat the microwell plate with VASP monoclonal antibody to make solid-phase antibody, divide the sample into two equal parts and treat them with activator and sample inhibitor respectively, add the VASP standard solution after sample treatment to the microwell coated with monoclonal antibody or sample solution, then add HRP-labeled phosphorylated VASP antibody to form antibody-antigen-enzyme-labeled antibody complex, after washing, add substrate TMB for color development; TMB is converted into blue under the catalysis of HRP enzyme, and in Un...

Embodiment 2

[0068] Embodiment 2, the test method of kit

[0069] (1) Take out the kit from the refrigerator and equilibrate to room temperature (18-25°C);

[0070] (2) Take sample activator dry powder and sample inhibitor dry powder and add 1ml sample pretreatment agent to dissolve;

[0071] (3) Take 10 μl sample activator and sample inhibitor respectively into the reaction well;

[0072] (4) Take 10 μl of the sample to be tested (whole blood) or the calibrator, add the sample activator and the sample inhibitor to the corresponding microwells of the microplate, mix well, and incubate at room temperature for 10 minutes;

[0073] (5) add to the corresponding microwell of the microplate, incubate, and carry out the first step reaction;

[0074] (6) Add HRP enzyme-labeled VASP monoclonal antibody, incubate in the dark, and carry out the second step of reaction;

[0075] (7) Wash, pat dry, add chromogenic solution, and incubate in the dark;

[0076] (8) Add stop solution to measure its abs...

Embodiment 3

[0078] Embodiment 3, the performance test of kit

[0079] (1) Use the kit of this application to pass the clinical sample verification

[0080] The kit of this application and the kits already on the market (CY-QUANTVASP / P2Y12, BIOCYTEX) detected 183 samples at the same time, and the data are shown in the following table:

[0081] The coincidence rate with the samples detected by Stago's VASP kit

[0082]

[0083] Negative coincidence rate: 84 / 88=95.45%;

[0084] Positive coincidence rate: 95 / 95=100%;

[0085] Total coincidence rate: 179 / 183=97.81%.

[0086] It can be seen from the above results that the human phosphorylated vasodilator-stimulated phosphoprotein ELISA kit of the present invention has good applicability and advancement.

[0087] (2) Linearity test of the kit

[0088] Draw the standard curve after using the protein calibrator to detect, the standard curve is as follows figure 1 , 2 As shown, the standard curve formula y (activation) = 1.1754x + 14.81, ...

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Abstract

The invention discloses an enzyme-linked immunoassay kit for human phosphorylated vasodilator-stimulating protein and a detection method thereof, wherein the detection kit includes a sample pretreatment buffer, a sample activator containing PGE1, and a PGE1-containing and ADP sample inhibitor, microtiter plate, VASP monoclonal antibody, chromogenic solution, and stop solution; the VASP monoclonal antibody is horseradish peroxidase-labeled mouse monoclonal antibody, and the heavy chain complementarity-determining region of the mouse monoclonal antibody contains CDR‑H1, CDR‑H2 and CDR‑H3 fragments, the complementarity determining region of the mouse monoclonal antibody light chain contains CDR‑L1, CDR‑L2 and CDR‑L3 fragments. The invention adopts ELISA, and optimizes and improves the sequence of the monoclonal antibody. The improved monoclonal antibody has a high capture rate and good specificity, overcomes the influence of whole blood samples on the test results, and eliminates the need for previous tests. Before that, the sample to be tested itself needs to be processed.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay kit for human phosphorylated vasodilator stimulating protein and a detection method thereof, belonging to the field of molecular immunology. Background technique [0002] Human phosphorylated vasodilator-stimulated phosphoprotein (VASP) belongs to the Ena / protein family and is a platelet intracellular actin regulatory protein. There are two important phosphorylation sites: Serine 157 and Serine 239. The phosphorylation status of these two phosphorylation sites is closely related to the activation function of platelets. Phosphorylation of the serine 157 phosphorylation site can inhibit the binding of fibrinogen and glycoprotein IIb / IIIa in human platelets; the serine 239 phosphorylation site mainly exists in intact human endothelial cells and platelets, and its phosphorylation responds to pharmacological and physiological vasodilators and platelet inhibitors. Vasodilator-stimulated phosphoprotein i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/543G01N21/31
CPCG01N33/6893G01N33/543G01N21/31
Inventor 刘丹易倩春
Owner 湖南菲思特精准医疗科技有限公司
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