Detection kit for detecting vascular endothelial growth factors as well as preparation method and use method thereof

A technology for detection kits and growth factors, applied in the field of detection kits, can solve the problems of low detection sensitivity, low accuracy, narrow detection range, etc., and achieve high sensitivity, improved accuracy and detection efficiency, and short reaction time. Effect

Pending Publication Date: 2020-04-21
SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current method used to detect human serum VEGF is mainly enzyme-linked immunosorbent assay, but its detection sensitivity is low, the detection range is narrow, the accuracy is not high, and the operation is relatively cumbersome.

Method used

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  • Detection kit for detecting vascular endothelial growth factors as well as preparation method and use method thereof
  • Detection kit for detecting vascular endothelial growth factors as well as preparation method and use method thereof
  • Detection kit for detecting vascular endothelial growth factors as well as preparation method and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of the vascular endothelial growth factor quantitative detection kit, the specific process is as follows: figure 1 .

[0039] (1) Preparation of biotinylated VEGF monoclonal antibody

[0040] Take 0.1 mg of VEGF monoclonal antibody, dilute it to 1 mg / mL with PBS buffer, weigh 1 mg of biotin and dissolve it in DMSO at a concentration of 10 mmol / L, mix according to the molar mass ratio of antibody and biotin 1:50, and store at room temperature Mix well for 30 minutes, then desalt with PD SpinTrap G-25 centrifugal desalting column. During the desalting process, first pretreat the desalting column with PBS, and finally add the obtained biotinylated VEGF monoclonal antibody solution, and collect the liquid in the centrifuge tube until storage. The biotinylated VEGF monoclonal antibody was obtained in the tube, added an equal volume of glycerol, mixed well, and stored at -20°C.

[0041] (2) VEGF monoclonal antibody labeled with alkaline phosphatase

[0042]...

Embodiment 2

[0046] Quantitative detection method of vascular endothelial growth factor

[0047] Using the automatic chemiluminescence immunoassay analyzer (Lumiart-II-1) as the detection tool, the detection principle is the double-antibody sandwich method. In a disposable cuvette, the instrument sequentially adds 50 μL of sample, 50 μL of biotinylated VEGF monoclonal antibody, and 50 μL of Alkaline phosphatase-labeled VEGF monoclonal antibody, reacted for 15 minutes, then added 25 μL streptavidin magnetic beads, reacted for 5 minutes, carried out magnetic separation, washed 5 times with cleaning solution, the mechanical arm sent the cuvette into the dark room, added 200 μL of chemiluminescence substrate solution (APS-5) was used for luminescence reaction, and finally the luminescence value was recorded.

Embodiment 3

[0049] Performance evaluation of vascular endothelial growth factor magnetic particle chemiluminescent immunoassay kit

[0050] Adopt the method in embodiment 2 to detect VEGF calibrator, draw standard curve such as figure 2 shown.

[0051] Detection of sensitivity: Refer to the recommended experimental scheme in CLSI EP17-A document, calculate the sensitivity of the vascular endothelial growth factor magnetic particle chemiluminescence immunoassay kit, and the obtained sensitivity is 2pg / mL.

[0052] Linear detection: for vascular endothelium with concentrations of 3.91pg / mL, 7.81pg / mL, 15.63pg / mL, 31.25pg / mL, 62.5pg / mL, 125pg / mL, 250pg / mL, 500pg / mL, 1000pg / mL The growth factor was analyzed linearly, the linear correlation coefficient was calculated, r=0.9996, and the linear detection range of the kit was 3.91-1000pg / mL.

[0053] Precision testing: Take the VEGF quality control product with a concentration of , do 10 parallels for each concentration, use three batches of k...

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Abstract

The invention relates to a detection kit for detecting vascular endothelial growth factors as well as a preparation method and a use method of the detection kit. The detection kit comprises a biotinylated VEGF monoclonal antibody, a VEGF monoclonal antibody labeled by alkaline phosphatase, and streptavidin magnetic beads. The kit is suitable for a full-automatic chemiluminiscence immunoassay analyzer Lumiart-II-1. Compared with a traditional ELISA kit, the kit reduces manual errors by means of fully automated operation, the reaction time is shorter, the sensitivity is higher, high-throughput rapid determination is achieved, the accuracy degree is high, the specificity is high, and the precision and detection efficiency are improved greatly; and the kit can be used for early cancer risk screening, tumor patient treatment effect evaluation monitoring so as to guide medication, recurrence monitoring, prognosis and the like, and has a good application prospect.

Description

technical field [0001] The invention relates to a detection kit, in particular to a detection kit for detecting vascular endothelial growth factor, a preparation method and a use method thereof. Background technique [0002] The growth, development and metastasis of tumor cells are multifactorial and multistage, involving the intricate relationship between tumor cells and hosts, and the mechanism is still unclear. Recent studies have found that the formation of new blood vessels is closely related to tumor development and metastasis. Vascular endothelial growth factor (vascular endothelial growth factor) is a highly biologically active functional signal transduction protein, which can act on vascular endothelial cells to make them proliferate, migrate, form tubes, participate in angiogenesis and make capillary Increased vascular permeability leads to abnormal growth of tumor blood vessels, hinders the effective delivery of anti-tumor drugs to tumor tissues, and stimulates t...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/574G01N33/543G01N33/535
CPCG01N33/6893G01N33/577G01N33/57484G01N33/54326G01N33/535G01N2333/475
Inventor 郭健健
Owner SHANGHAI FOSUN LONG MARCH MEDICAL SCI CO LTD
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