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Biological activity measurement method for fibronectin

A fibronectin, biologically active technology, used in biological testing, color/spectral property measurement, material testing, etc., can solve the problem of large error in results, concealed epitope exposure due to conformational changes, and cannot directly reflect the mechanism of action and activity, etc. problem, to achieve the effect of quick acquisition and simple experimental steps

Inactive Publication Date: 2019-03-29
NOVOPROTEIN SCI INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process is initiated by binding of fibronectin to cell surface integrins, resulting in conformational changes and exposure of cryptic epitopes required for aggregation
[0003] The existing biological activity detection method for fibronectin (Fn) is to detect the affinity between fibronectin (Fn) and gelatin. This method has a large error in the results and cannot directly reflect the mechanism and activity of the protein in vivo.

Method used

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  • Biological activity measurement method for fibronectin
  • Biological activity measurement method for fibronectin

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Effect test

Embodiment 1

[0032] Embodiment 1: the biological activity assay method of fibronectin (Fn)

[0033] According to the principle of cell adhesion, the invention wraps the sample to be tested, adds Jurkat cells, and uses the CCK-8 method to detect the amount of attached cells, so as to reflect the activity of the sample to be tested.

[0034] 1. Cell recovery: Take out the frozen Jurkat cells from the liquid nitrogen tank, place the cell cryopreservation tube in a 37°C water bath, and guide the liquid in the cryopreservation tube to dissolve completely. The liquid in the freezing tube was mixed with 5 ml of 1640 medium containing 10% FBS, and centrifuged at 850 rpm for 5 min. Discard the supernatant in the centrifuge tube, resuspend the cell pellet with 6ml of 10% FBS 1640 medium, resuspend and mix well, and add the cells into a T25 cell culture flask.

[0035] 2. Cell passage: Jurkat cells were counted and passaged every other day, and the passaged cell density was 3×10 5 cells / ml, and the...

Embodiment 2

[0042] Embodiment 2: the biological activity assay result analysis of fibronectin (Fn)

[0043] After the microplate reader detects, the reading value of the 96-well plate:

[0044]

[0045] Analyze the obtained values ​​by drawing analysis software Origin8 and the results are as follows: figure 1 :

[0046] Depend on figure 1 It can be seen from the results that as the concentration of cladding protein changes, the number of Jurkat cells adhered to cladding protein also changes, and it shows an increasing trend with the increase of cladding protein concentration. A complete S-shaped curve can be obtained through software drawing, and the value of the half effective dose base ED50 of fibronectin (Fn) can be obtained as 421.3 ng / ml through the analysis software.

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Abstract

The invention provides a biological activity measurement method for fibronectin. The biological activity measurement method for the fibronectin comprises the steps that a plate is coated with the fibronectin; cells are added for adhesion; the quantity of the adhered cells is measured; and then the biological activity is calculated. According to the method, biological activity detection is conducted on the fibronection by adopting the adhesion effect between the cells and the protein; and the created detection method can accurately, quickly and conveniently detect the biological activity of thefibronectin (Fn) and can be widely applied to biological activity detection on the fibronectin (Fn). The method has the following advantages of operability, convenience and accuracy.

Description

technical field [0001] The invention belongs to the field of biological products, in particular to a method for detecting the biological activity of fibronectin. Background technique [0002] Fibronectin (Fn) is a large, modular glycoprotein found as a network of polymer fibers in the extracellular matrix (ECM) and as a soluble disulfide-linked dimer in plasma and other body fluids. The protein subunits consist of three types of homologous structural repeats, known as Fn type I, type II and type III. Fibronectin is a ligand for fibrin, heparin, chondroitin sulfate, collagen / gelatin and many integrin receptors. It is involved in a variety of cellular processes including cell adhesion / migration, blood coagulation, morphogenesis, tissue repair and cell signaling. Fibronectin function is mediated by insoluble polymeric fibrils in the ECM. The conversion of non-functional soluble fibronectin to fibronectin fibrils is tightly regulated in the ECM. The process is initiated by b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N21/31
CPCG01N21/31G01N33/68
Inventor 由超朱梦洁王笃强
Owner NOVOPROTEIN SCI INC
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