Affinity particle and affinity separation method

a technology of affinity separation and affinity particles, which is applied in the field of affinity particles and affinity separation methods, can solve the problems of large quantity of sample, poor purification efficiency, and time-consuming purification, and achieves superior dispersion properties, high separation selectivity, and suppression of adsorption of other substances.

Inactive Publication Date: 2007-08-09
SHISEIDO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0026] The affinity particles of the present invention use ligands to capture only a certain target substance (the substance desired to be separated) and suppresses adsorption of other substances onto the particles, resulting in a very high separation selectivity. They also exhibit superior dispersion properties and make separation from liquid samples very easy, which makes it possible to separate the target substance easily and with high accuracy by using inexpensive inorganic powder particles for affinity particles.
[0027] That is, the target substance separation method of the present invention can effectively and easily separate the target substance to be separated in a short amount of time. Since substances have a tendency to adsorb onto foreign substances, conventional affinity particles have difficulties efficiently isolating only the target substance; however, it is possible to very efficiently prevent non-specific adsorption of other substances to the affinity particles and thus increase the purification yield by modifying the particle surface with phosphorylcholine groups.
[0028] Also, phosphorylcholine groups are extremely hydrophilic and they also improve the dispersion properties of the affinity particles in a liquid sample containing water.
[0029] Furthermore, conventional particles tend to aggregate in the presence of salts and therefore the purification efficiency decreases when the target substance is to be isolated from serum because of aggregation due to various salts in serum; however, the affinity particles of the present invention don't aggregate significantly even under the presence of salts, which makes it possible to recover the target substance efficiently.
[0030] The affinity particles used in the present invention are composed of inorganic particles and therefore their specific gravity is high, which makes it easy to recover them by leaving the mixture at rest or by light centrifugation. These particles can be filled in a column as carriers and such a column can be used as an affinity column to recover a target substance.

Problems solved by technology

However, column separation has some fatal problems as described in the following (1) to (3): (1) Many kinds of columns have to be used to obtain the target substance, resulting in a poor purification efficiency.
(2) A verification test is required to make sure the target substance is contained in the fractionated ingredients, which means purification is time consuming.
(3) Because of the large purification loss, a large quantity of the sample is required.
However, separation and purification using affinity columns have the following problems: (1) The desired target substance is not selectively separated.
(2) The capture efficiency is low, which means a large quantity of the liquid sample is required.
The affinity separation method in which affinity particles are dispersed in a liquid sample for separation uses agarose and such (Non-patent Document 1), but this method has the following problems: (1) The desired target substance is not selectively separated.
(2) The specific gravity is small, which makes the separation of the affinity particles difficult.
(3) The carrier is easily disintegrated, which leads to poor durability.
On the other hand, inorganic particles adsorb more substances than organic particles do, therefore those skilled in the art didn't think of using inorganic particles for affinity particles.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

synthesis example 1

“An aldehyde Chemical Compound Containing Phosphorylcholine Groups”

[0135] 1-alpha-glycerophosphorylcholine (6.29 g) was dissolved in 210 ml of distilled water and cooled in an ice water bath. Sodium periodate (10.23 g) was added, followed by five hours of stirring. The reaction fluid was concentrated under reduced pressure and dried under reduced pressure; methanol was then used to extract the target substance. The structure is shown in the following chemical formula (6).

[0136] A 1H NMR spectrum of the compound of formula (6) is shown in FIG. 2. Since the compound of formula (6) is in equilibrium with formula (9) in water, the actual spectrum reflects both formula (6) and formula (9).

synthesis example 2

“A Carboxylic Acid Chemical Compound Containing Phosphorylcholine Groups”

[0137] 5 g of glycerophosphorylcholine, 17 g of sodium periodate, 81 mg of ruthenium trichloride n-hydrate, 70 g of ion-exchanged water and 30 g of acetonitrile were put into a 200 ml flask. After stirring for two hours at room temperature, filtering was carried out and the solvent was removed from the filtrate. Methanol was used to extract the target compound from the obtained solid product; methanol was then removed to obtain the target compound (7).

[0138] A 1H NMR spectrum of the compound of formula (7) is shown in FIG. 3.

synthesis example 3

“Compound of Formula (10)”

[0139] 9.0 g of the compound of Synthesis example 1 was dissolved in 55 mL of dehydrated methanol, and the air inside the vessel was replaced by dry nitrogen. Next, 2.84 g of 3-aminopropyltrimethoxysilane was added to the methanol solution of chemical compound 1. This mixed solution was stirred overnight at room temperature and cooled with ice; 1.39 g of sodium cyanohydroborate was then added and the temperature was returned back to room temperature, followed by 5 hours of stirring. During this time dry nitrogen was continued to be fed through the reaction vessel. After filtering the precipitation, a methanol solution of the target substance, i.e. the compound of the following formula (10), was obtained.

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Abstract

The present invention is affinity particles that are characterized by having phosphorylcholine groups represented by the following formula (1) covalently bonded onto the surface of inorganic powder and also by having ligands having specific affinity with a certain target substance covalently bonded or adsorbed onto the surface of inorganic powder. The object of the present invention is to provide an affinity separation method that uses affinity particles utilizing inexpensive inorganic particles and is capable of separating the target substance easily and with high accuracy.

Description

TECHNICAL FIELD [0001] The present invention relates to affinity particles and an affinity separation method. More specifically, it relates to affinity particles utilizing inorganic particles and an affinity separation method that allows easy and highly precise separation of the target substance. The present invention is very useful in various separation, purification, and testing methods including latex agglutination methods and immunoprecipitation methods that allow easy and highly sensitive detection of the target substance. BACKGROUND ART [0002] Conventionally, column chromatography has been used for separation and purification of biological substances. However, column separation has some fatal problems as described in the following (1) to (3): [0003] (1) Many kinds of columns have to be used to obtain the target substance, resulting in a poor purification efficiency. [0004] (2) A verification test is required to make sure the target substance is contained in the fractionated in...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): B01D15/08B01D15/38B01J20/22B01J20/286B01J20/32C07F9/09
CPCB01J20/3204B01J20/3285B01J20/286B01J2220/58B01J20/3248B01J20/3259B01J20/3274B01J20/3219Y10T428/2982G01N33/54393B01J20/28004B01J20/3242C07F9/091B01D15/3804C07F9/02C07F9/06C07F9/09
Inventor MAENO, KATSUYUKIMIYAZAWA, KAZUYUKIISHIKUBO, AKIRAISHIHARA, KAZUHIKO
Owner SHISEIDO CO LTD
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