Detection method of collagen terminal peptide

A detection method, collagen technology, applied in the field of collagen detection, can solve problems such as inapplicability, complexity, and false negatives, and achieve the effect of improving the range of detection capabilities

Inactive Publication Date: 2018-11-23
GUANGZHOU TRAUER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex system of extracted exogenous collagen or decellularized product collagen (for example, the extracted collagen system contains enzymes, enzyme-cleaved telopeptides, miscellaneous proteins, etc.; another example is that the decellularized product collagen contains other cells Extracellular matrix;), will interfere with the detection, easily cause false positive, false negative and other results, poor stability
Therefore, these methods are not suitable for judging the content of extracted collagen telopeptides

Method used

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  • Detection method of collagen terminal peptide
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  • Detection method of collagen terminal peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Detection of bovine type I collagen C-terminal antigen-specific band

[0042] One embodiment of the detection method of the collagen peptide telopeptide of the present invention comprises the following steps:

[0043] Sample processing: 5mL test sample (collagen containing telopeptide, such as figure 1 Collagen, collagen means collagen), 5mL negative control sample (collagen with telopeptide removed) were mixed with 5mL SDS buffer respectively, treated in boiling water bath at 100°C for 3min, cooled to room temperature, at 4°C, 5000rmp Centrifuge for 5 minutes to obtain the loading sample for later use; configure 6.5% separation gel and 4% stacking gel; the loading volume of each well is 10uL, and run at a current of 10mA for 30min. After the bromophenol blue in the sample enters the separation gel, adjust the electrophoresis The current is 20min.

[0044] Transfer: equilibrate the nitrocellulose membrane, 6 pieces of filter paper and the recovered gel in th...

Embodiment 2

[0048] Example 2 Excision effect test of bovine type I collagen C-terminal and N-terminal under different extraction time

[0049] Sample description: Take about 200g of beef tendon, add 15g of pepsin, concentration of 0.5M, volume of 1L acetic acid solution, stir evenly, let it stand at 4°C for 14h, then place it in an electric mixer and stir at 4°C After extraction for 96 hours, the extracted collagen mixture was centrifuged at a speed of 10000rmp for 20 minutes at intervals, and used for western blot (ie westernblot) analysis.

[0050] One embodiment of the detection method of the collagen peptide telopeptide of the present invention comprises the following steps:

[0051] Sample processing: mix 5mL of the sample to be tested at the sampling time point with 5mL of SDS buffer, treat it in a boiling water bath at 100°C for 3min, cool to room temperature, and centrifuge at 5000rmp at 4°C for 5min to obtain the loaded sample for later use; configuration 6.5 % separation gel an...

Embodiment 3

[0058] Example 3 Detection of bovine type I collagen sponge N-terminal antigen-specific band

[0059] Sample description: Weigh three batches of collagen sponges of 0.05±0.0005g each, add 10mL sample buffer solution (100mM Tris, 8M urea, 2% SDS, 2% mercaptoethanol, pH8.0 ) mixing, at room temperature, shaking and dissolving for 4 hours, centrifuging at 5000rmp for 5 minutes, and taking the supernatant for later use.

[0060] One embodiment of the detection method of the collagen peptide telopeptide of the present invention comprises the following steps:

[0061] Sample processing: Mix 5mL of supernatant with 5mL of SDS buffer and treat it in a boiling water bath at 100°C for 3min. After cooling to room temperature, centrifuge at 4°C and 5000rmp for 5min to obtain the loading sample for later use; configure 6.5% separating gel and 4% stacking gel; the loading volume of each hole is 10uL, run at a current of 10mA for 30min, after the bromophenol blue in the sample enters the se...

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Abstract

The invention discloses a detection method of a collagen terminal peptide. The detection method comprises the following steps: (1) performing treatment and electrophoresis on a sample; (2) protein transfer printing; (3) closing and incubating: oscillating a transfer printing film obtained in step (2) into a closing solution; adding a collagen terminal peptide antibody to performing the primary antibody incubation for the transfer printing film, and then adding a secondary antibody to perform the incubation; and (4) performing the color developing reaction: adding the transfer printing film after the secondary antibody incubation of step (3) into a color developing solution to develop until a stripe appears, then adding distilled water to terminate the color developing reaction, and judgingthe type of the collagen terminal peptide in a to-be-detected sample according to the stripe. The invention provides the method which can effectively qualitatively and half-quantitatively detect thecontent of terminal peptide in exogenous collagen, and provides method evidence for the immunogenicity of an exogenous collagen-based biological product.

Description

technical field [0001] The invention relates to the technical field of collagen detection, in particular to a detection method for collagen telopeptides. Background technique [0002] Exogenous collagen-based organisms play a huge role in hemostasis after tissue trauma, repair, defect filling, and regenerative medicine developed for tissue engineering scaffolds. Although collagen is generally considered to be a safe and multifunctional biomaterial, with the increasing application of collagen in tissue engineering, the possible clinical immune response of collagen has attracted much attention. The antigenic determinants of exogenous collagen are mainly divided into three categories: ①The antigenic determinants of the complete helical structure, which have weak immunogenicity; Immunogenicity is shown only after the structure is destroyed; ③C- and N- (collectively referred to as collagen telopeptides) epitopes, the immunogenicity of this part is considered to be the biggest ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N2333/78
Inventor 梁健华赖俏荣李奕恒
Owner GUANGZHOU TRAUER BIOTECH
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