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Separation method and analysis method of osteopontin

An osteopontin and separation method technology, applied in the field of osteopontin separation and analysis, can solve the problems of inaccurate analysis results, high cost, complicated operation, etc., and achieve the effects of simple and fast operation, low cost, and accurate analysis

Pending Publication Date: 2021-10-29
HEILONGJIANG FEIHE DAIRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The main purpose of the present invention is to provide a method for separating and analyzing osteopontin, so as to solve the problems of complicated operation, high cost and inaccurate analysis results in the analytical method of osteopontin in the prior art

Method used

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  • Separation method and analysis method of osteopontin
  • Separation method and analysis method of osteopontin
  • Separation method and analysis method of osteopontin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1) Instruments and conditions:

[0052] High performance liquid chromatography: ACQUITY Arc Bio UHPLC system, UV detector;

[0053] Chromatographic column: Waters Protein-Pak Hi Res Q, packing material particle size 5μm, 4.6×100mm, P / N: 186004931;

[0054] Column temperature: 35°C;

[0055] Wavelength: 214nm;

[0056] Needle washing solution: 0.1% TFA aqueous solution;

[0057] Injection volume: 5 μL;

[0058] Mobile phase: A: an aqueous solution containing 20mmol / L Tris (trishydroxymethylaminomethane solution) and 10mmol / L NaCl at pH=8.0; B: an aqueous solution containing 20mmol / L Tris and 800mmol / L NaCl at pH=8.0.

[0059] Gradient elution program:

[0060] Table 1

[0061] Time(min) Flow rate mL / min ％A ％B 1 0 0.4 50 50 2 3 0.4 20 80 3 5 0.4 10 90 4 7 0.4 10 90 5 7.5 0.4 50 50 6 15 0.4 50 50

[0062] Add OPN to the basic milk powder, and detect the chromatographic peak position of OPN under the...

Embodiment 2

[0082] The test object is to add OPN to the basic formula milk without adding OPN, and the content of OPN is 0.1mg / L. The test process of embodiment 2 is the same as that of embodiment 1.

Embodiment 3

[0084] The test object is the same as in Example 2.

[0085] The difference between the test process of embodiment 3 and embodiment 1 is that the chromatographic column is selected as Thermo FisherScientific ProPac WAX-10, P / N: 054999; the injection volume is 10 μL; the gradient program is:

[0086] Table 2

[0087] Time(min) Flow rate mL / min ％A ％B 1 0 1.0 50 50 2 7 1.0 10 90 3 10 1.0 10 90 4 10.5 1.0 50 50 5 20 1.0 50 50

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Abstract

The invention provides a separation method and an analysis method of osteopontin. The separation method comprises the following steps: carrying out HPLC separation on a to-be-tested substance containing osteopontin by adopting a strong anion exchange chromatographic column to obtain an osteopontin sample, wherein a filling material in the strong anion exchange chromatographic column comprises surface porous silica gel bonded with trimethyl aminopropyl, or a microporous ethyl vinyl benzene cross-linked 55% divinyl benzene polymer bonded with trimethyl aminopropyl. According to the invention, the strong anion exchange chromatographic column used in the invention achieves the purpose of effectively separating OPN from other impurities; and compared with an analysis method for analyzing the osteopontin peptide fragment by adopting mass spectrometry in the prior art, the separation method of OPN in the invention has the advantages that the retention difference of each component in the to-be-tested object containing OPN on a chromatographic column is obvious, the selectivity and the sensitivity on OPN are generally excellent, and the separation capability is better, so that the OPN in the to-be-tested object can be analyzed more accurately.

Description

technical field [0001] The invention relates to the technical field of osteopontin analysis, in particular to a method for separating and analyzing osteopontin. Background technique [0002] Osteopontin (OPN) is an acidic phosphorylated glycoprotein that has multiple functions including maintaining intestinal integrity by affecting tight junctions, which help maintain the intestinal mucosal barrier, maintain intestinal structure, regulate intestinal Inflammation, cell adhesion, bone remodeling, angiogenesis, regulation of calcium oxalate formation, and cell signaling. OPN has been implicated in human immune responses, including opsonin activity in mouse models and contributing to inflammatory cell migration to wound sites and cytokine expression. Senger et al. identified OPN in breast milk and subsequently in milk, albeit at lower levels. In breast milk, OPN accounts for about 100-300 mg / L of the total protein, with an average of 138 mg / L. Bovine milk contains about 15–20...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/74G01N30/96
CPCG01N30/02G01N30/74G01N30/96Y02A50/30
Inventor 蒋士龙王象欣解庆刚刘莹崔东影梁爱梅冷友斌
Owner HEILONGJIANG FEIHE DAIRY