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Method for determination of molecular weight of hyaluronic acid (патент US № 8163498)

Официальная публикация
патента US № 8163498

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Классы МПК:G01N33/53 ...иммунологический анализ, анализ биоспецифического связывания, материалы для этого
Автор(ы):Fujita, Hiroshi (Higashiyamato, JP)
Патентообладатель(и):Seikagaku Corporation (Chiyoda-ku, Tokyo, JP)
Приоритеты:
подача заявки
08.08.2007
публикация патента
24.04.2012

РЕФЕРАТ (Abstract)

A method of measuring a molecular weight of hyaluronic acid, comprising at least reacting hyaluronic acid in a sample containing the hyaluronic acid with a hyaluronic acid-binding protein to measure an amount of the hyaluronic acid-binding protein that is bound to the hyaluronic acid in the sample or a value that reflects the amount.

ФОРМУЛА ИЗОБРЕТЕНИЯ (CLAIMS)

What is claimed is:

1. A method of measuring a molecular weight of hyaluronic acid, comprising at least the following steps (1) to (3): (1) measuring a concentration of hyaluronic acid in a sample; (2) reacting hyaluronic acid in a sample containing the hyaluronic acid with a hyaluronic acid-binding protein, and measuring an amount of the hyaluronic acid-binding protein that is bound to the hyaluronic acid in the sample or a value that reflects the amount; and (3) determining the molecular weight of the hyaluronic acid in the sample from the concentration of the hyaluronic acid in the sample obtained in step (1); and the amount of the hyaluronic acid-binding protein that is bound to the hyaluronic acid in the sample or the value that reflects the amount obtained in step (2), wherein the molecular weight in the sample is determined from a relationship between the molecular weight in a standard hyaluronic acid sample with known concentration and molecular weight and the amount of the hyaluronic acid-binding protein binding to the standard hyaluronic acid sample or the value that reflects the amount.

2. The method according to claim 1, wherein step (2) comprises the following steps (i) to (iv): (i) immobilizing the hyaluronic acid-binding protein to a solid phase; (ii) reacting the hyaluronic acid-binding protein that is immobilized to the solid phase with the hyaluronic acid in a sample; (iii) further reacting the hyaluronic acid that is bound to the hyaluronic acid-binding protein that is immobilized to the solid phase with a labeled hyaluronic acid-binding protein; and (iv) measuring an amount of the labeled hyaluronic acid-binding protein that is bound to the hyaluronic acid in step (iii) or a value that reflects the amount.

3. The method according to claim 2, wherein a label substance of the labeled hyaluronic acid-binding protein to be used in step (iii) is selected from the group consisting of biotin, avidin, an enzyme, an isotope, a fluorescent pigment, and a chemiluminescent substance.

4. The method according to claim 2, wherein a label substance of the labeled hyaluronic acid-binding protein to be used in the step (iii) is biotin or avidin.

5. The method according to claim 1, wherein the relationship between the molecular weight of the hyaluronic acid and the amount of the hyaluronic acid-binding protein binding to the hyaluronic acid or the value that reflects the amount, the relationship being used in step (3) and obtained from the standard hyaluronic acid sample with known concentration and molecular weight, is represented by a standard curve that is obtained by performing step (2) using the hyaluronic acid standard solution with known concentration and molecular weight as the sample.

6. The method according to claim 1, wherein, in step (3), the concentration of the hyaluronic acid in the sample that is obtained in step (1) is used as an index for dilution of the sample or used for calculation for the amount of the hyaluronic acid-binding protein binding to the hyaluronic acid in the standard hyaluronic acid sample at the concentration or the value that reflects the amount.

7. The method according to claim 2, wherein, in reacting the hyaluronic acid with the hyaluronic acid-binding protein, the amount of the hyaluronic acid-binding protein that is bound to the hyaluronic acid is adjusted by allowing an additive selected from a protein denaturant, an acidic polysaccharide, and a surfactant to coexist and regulating an amount of the additive.

8. The method according to claim 2, wherein the amount of the hyaluronic acid-binding protein that is bound to the hyaluronic acid or the value that reflects the amount to be measured in step (iv) is regulated by changing the amount of the hyaluronic acid to be immobilized in step (i) or the amount of the hyaluronic acid-binding protein to be reacted in step (iii).

9. The method according to claim 7, wherein the additive comprises guanidine hydrochloride.

10. The method according to claim 2, wherein the amount of the hyaluronic acid-binding protein to be immobilized is 3to 30,000 ng/mL as the concentration of a solution of the hyaluronic acid-binding protein to be used for the immobilization.

11. The method according to claim 2, wherein: the labeled hyaluronic acid-binding protein to be reacted in step (iii) comprises a biotin-labeled hyaluronic acid-binding protein; and a solution of the biotin-labeled protein has a concentration of 10 to 30,000 ng/mL.

12. The method according to claim 9, wherein guanidine hydrochloride in the hyaluronic acid standard solution and/or the sample have/has a concentration up to 3.6 M at a final concentration.

13. The method according to claim 9, wherein guanidine hydrochloride in the solution of the biotin-labeled hyaluronic acid-binding protein has a concentration up to 1.6 M at a final concentration.


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