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A photoactivated chemiluminescence homogeneous immunoassay kit for human phosphorylated vasodilator-stimulated phosphoprotein and its detection method

A light-induced chemiluminescence and vasodilation technology, applied in the field of molecular immunology, can solve the problems of narrow linear range, poor stability, cumbersome operation, etc., and achieve the effect of wide detection range, simple instrument cost, and good repeatability

Active Publication Date: 2021-09-28
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

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Problems solved by technology

[0008] In view of the above-mentioned problems existing in the prior art, the object of the present invention is to obtain a photoactivated chemiluminescence homogeneous immunoassay kit and a detection method for human phosphorylated vasodilator-stimulated phosphoprotein, which avoids the linearity of the enzyme-linked immunoassay kit. Narrow range, cumbersome operation and other shortcomings, and solve the defects of low sensitivity and poor stability

Method used

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  • A photoactivated chemiluminescence homogeneous immunoassay kit for human phosphorylated vasodilator-stimulated phosphoprotein and its detection method
  • A photoactivated chemiluminescence homogeneous immunoassay kit for human phosphorylated vasodilator-stimulated phosphoprotein and its detection method
  • A photoactivated chemiluminescence homogeneous immunoassay kit for human phosphorylated vasodilator-stimulated phosphoprotein and its detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the preparation of kit

[0040] The kit of the invention adopts homogeneous immunization, utilizes a light-induced chemiluminescence analysis method and cooperates with a light-induced chemiluminescence detector, and is used for measuring the content of human phosphorylated vasodilator-stimulated phosphoprotein in a human body sample. The technical principle of the reaction is: by mixing the sample, biotin-labeled VASP-P antibody I and luminescent microspheres coupled with VASP-P antibody II under homogeneous conditions. At this time, the biotin-labeled VASP-P antibody I and the luminescent microsphere coupled with the VASP-P antibody II can quickly and effectively recognize the target molecule of the detection sample to form an immune sandwich complex. Then add streptavidin-labeled photosensitive microspheres to form photosensitive microspheres-streptavidin-biotin-VASP-P antibody I-VASP-P antigen-VASP-P antibody II-luminescent microsphere immune complex ...

Embodiment 2

[0108] Embodiment 2, the test method of kit

[0109] (1) Take out the kit from the refrigerator and equilibrate to room temperature (18-25°C);

[0110] (2) Take sample activator dry powder and sample inhibitor dry powder and add 1ml sample pretreatment agent to dissolve;

[0111] (3) Take 10 μl sample activator and sample inhibitor respectively in the reaction well;

[0112] (4) Take 10 μl of the sample to be tested (whole blood) or calibrator and add it to the sample activator and sample inhibitor reaction wells, mix well, and incubate at room temperature for 10 minutes;

[0113] (5) Add 20ul component FG-Ab and 20ul component Bio-Ab in turn to the reaction well, mix thoroughly, and incubate at 37°C for 10min;

[0114] (6) Add 175ul component SA-GG and incubate at 37°C for 2min;

[0115] (7) Laser irradiates microwells and calculates the signal value of each well;

[0116] (8) Calculation: platelet response index (PRI) = [(RLU activation - RLU inhibition) / (RLU activatio...

Embodiment 3

[0118] Embodiment 3, the performance test result of kit

[0119] Kit performance evaluation includes the determination of linearity, precision and accuracy of the kit prepared by this method.

[0120] (1) Draw a standard curve after detection with protein calibrator, and activate the standard curve as figure 2 As shown, the inhibition standard curve is as image 3 As shown, the standard curve formula y (activation) = 117.66x - 926.74, R2 = 0.9995, y (inhibition) = 114.65x + 101.47, R2 = 0.9997 At the same time, the linearity of the test results is evaluated, and the test results are shown in Table 1 below:

[0121] Table 1 Test results of calibrator

[0122]

[0123] (2) Test repeatability:

[0124] Table 2 Reproducibility of samples detected by VASP photo-activated chemiluminescence kit

[0125]

[0126] From the above test results and analysis, it can be seen that the test results of the kit of the present invention have good repeatability, and the results are all...

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Abstract

The invention belongs to the technical field of immune analysis, and in particular relates to a human phosphorylated vasodilator-stimulated phosphoprotein (VASP-P) photo-activated chemiluminescence homogeneous immunoassay kit and a detection method thereof, wherein the kit includes component SA ‑GG, Component Bio‑Ab, Component FG‑Ab, Sample Pretreatment Buffer, Sample Activator with PGE1 and Sample Inhibitor with PGE1+ADP. The kit of the present invention adopts homogeneous immunization, utilizes the light-induced chemiluminescence analysis method to cooperate with the light-induced chemiluminescence detector, and is used to measure the content of human phosphorylated vasodilator-stimulated phosphoprotein in human body samples, which is in line with the current mainstream technology of VASP detection. Compared with ELISA, it has simpler requirements for operators and lower instrument cost. Compared with ELISA and other heterogeneous immunoassays, it has a wide detection range, simple operation, short detection time, high sensitivity and good repeatability.

Description

technical field [0001] The invention relates to a photo-activated chemiluminescence homogeneous immunoassay kit and a detection method for human phosphorylated blood vessel expansion stimulating phosphoprotein, belonging to the field of molecular immunology. Background technique [0002] Human phosphorylated vasodilator-stimulated phosphoprotein (VASP) belongs to the Ena / VASP 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 inhibi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/531G01N33/58G01N21/76
CPCG01N21/76G01N33/531G01N33/577G01N33/587G01N33/6893G01N2333/47G01N2800/32
Inventor 刘丹陈立波
Owner 湖南菲思特精准医疗科技有限公司
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