JP2002214235A - Hyaluronic acid measuring method and kit for measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve problems such as a reduction in the effects of agitation during analysis due to the deposition, high specific gravity, and high viscosity of a crystal caused by a conventional hyaluronic acid measuring method which requires a high salt concentration. SOLUTION: A surface active agent is added to a buffer solution used for measuring hyaluronic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel method for measuring hyaluronic acid and a kit for measurement used in the method.

[0002]

2. Description of the Related Art Hyaluronic acid is an acidic mucopolysaccharide in which N-acetylglucosamine and glucuronic acid are alternately polymerized by β-1,4 bonds, has an action of bonding subcutaneous tissues, and has an action of umbilical cord and joint cavity. It is contained in synovial fluid and the vitreous body of the eye. It has been known that the concentration of hyaluronic acid in blood increases due to diseases such as liver fibrosis, inflammation such as rheumatism, or bone metastasis of cancer (Takato Ueno et al., Gastroenterology 105, 47).
5-481, 1993; E Laurent et al.
Annals of the Rheumatic Di
seases 44, 83-88, 1985;
Delpech et al. Analytical Bioche
mystery 149, 555-5651985).
Therefore, it can be said that quantifying hyaluronic acid in blood is useful for diagnosis of progression of liver fibrosis or inflammation, recovery after surgery, or diagnosis of cancer. As a method of measuring the concentration of hyaluronic acid in blood, a sandwich binding protein assay using a binding protein in cartilage has already been developed, and is currently widely used for in vitro diagnosis.
No. 41952, Koji Kondo et al. Clinical Pathology Vol. 39, No. 5, 536-540, Mayumi Hikosaka et al. Clinical Laboratory Instruments / Reagents, Vol. 18, No. 6, 984-988).

[0003]

In this measurement of blood hyaluronic acid, most of the sample, hyaluronic acid, forms proteoglycan in tissues in a living body and binds to proteins and carbohydrates in serum. When performing a binding protein assay, accurate measurement of hyaluronic acid cannot be performed unless the bond is dissociated. As a dissociation method, a method of adding an ionic compound to a buffer solution to increase the ionic strength (salt concentration) during the reaction is already known, and is also applied to this method. However, since the concentration added to the buffer solution is as high as 1.0 to 2.0 M, there are problems such as precipitation of crystals and reduction of the stirring effect during analysis due to increase in specific gravity and viscosity. .

[0004]

The inventors of the present invention have made intensive studies, and as a result, firstly, as a base for lowering the salt concentration to reduce the specific gravity, and for further promoting the dissociation of hyaluronic acid in serum. It has been found that the above problem can be solved by adding a surfactant, and the present invention has been completed based on this finding.

That is, the present invention provides a method for measuring hyaluronic acid, which comprises using a surfactant in a measurement system. The present invention also provides a kit for measuring hyaluronic acid, which contains a surfactant and a reagent necessary for measurement.

[0006] The type of surfactant used in the measurement method of the present invention is not particularly limited, but nonionic surfactants can be used, and are preferably polyethylene glycol type nonionic surfactants. It is preferable to include one or more of oxyethylene alkylphenyl ethers.

Embedded image (In the formula, R is a linear or branched alkyl group having 1 to 12 carbon atoms, and n is an integer of 30 to 100.) Examples of the alkyl group include a methyl group, an ethyl group, a linear group, Branched propyl, straight or branched butyl, straight or branched pentyl, straight or branched hexyl, straight or branched heptyl, straight or branched octyl A linear or branched nonyl group, a linear or branched decyl group, a linear or branched undecyl group, and a linear or branched dodecyl group. When there are two or more branched chains, that is, when n is 4 or more, any of the branched chains may be used. Preferred examples of the alkyl group include an octyl group and a nonyl group.

The method for measuring hyaluronic acid used in the present invention may be any method as long as it has been conventionally used, and is not particularly limited. Examples thereof include a solid phase method, a competitive method, and a binding method based on a binding protein assay. An agglutination method, a turbidimetric method and the like can be mentioned.

In the present invention, serum, plasma, synovial fluid, pleural effusion, tissue extract, culture supernatant, or urine can be used as a sample from which hyaluronic acid can be measured.

The measuring method according to the present invention will be described below by taking a sandwich binding protein assay as an example. According to the sandwich binding protein assay, a buffer containing a surfactant is added to the reaction solid phase, and a sample containing the sample hyaluronic acid is added to the hyaluronic acid-binding protein fixed to the solid phase, and The sample hyaluronic acid and the sample hyaluronic acid, and further adding the hyaluronic acid-binding protein and a labeling substance, respectively, or adding a hyaluronic acid-binding protein previously labeled with a labeling substance, Forming a sandwich-like complex between the hyaluronic acid-binding protein fixed on the solid phase, the sample hyaluronic acid and the labeled hyaluronic acid-binding protein, Is quantified.

Specifically, first, a hyaluronic acid-binding protein is fixed on a surface of a reaction plate on a solid phase. On this occasion,
On the solid phase, there is a surface portion on which the hyaluronic acid-binding protein or other molecular species can be fixed, and other blood components contained in the sample to be measured are added when the sample to be measured is added. There is fear. For this reason, it is preferable to add a block before adding the sample to cover the portion where the hyaluronic acid-binding protein is not fixed. As such a block body, for example, γ-
Examples include globulin, serum albumin, and serum. When a polystyrene plate is used as a solid phase, bovine serum albumin is preferred.

Next, a buffer containing a surfactant is added to the solid phase to which the hyaluronic acid-binding protein is fixed, and at the same time, a sample containing a high molecular weight hyaluronic acid is added. At this time, the sample does not need to have the high molecular weight hyaluronic acid extracted and separated, and blood or body fluid of a human body or the like can be used as it is. Alternatively, the sample may be dissolved in a block solution or diluted serum and added to the solid phase to which the hyaluronic acid-binding protein is fixed. In the measurement of hyaluronic acid, it is known that most of the hyaluronic acid contained in the sample forms proteoglycan in the tissue in the living body, and is bound to proteins and carbohydrates in serum. When performing the method, accurate hyaluronic acid cannot be measured unless the bond is dissociated. In the measurement method of the present invention, hyaluronic acid can be added without increasing the ionic strength (salt concentration) of the ionic compound by adding a surfactant such as polyoxyethylene alkylphenyl ether to the buffer together with the ionic compound as a base. Can be dissociated from proteins, saccharides, etc. After binding the high-molecular-weight hyaluronic acid to the hyaluronic acid-binding protein fixed on the solid phase in this manner, it is preferable to wash the surface of the solid phase.

Further, a hyaluronic acid-binding protein, which has been labeled with a predetermined labeling substance, is added to the solid phase to which the high molecular weight hyaluronic acid is bound. Examples of the labeling substance include isotopes, fluorescent dyes, avidin, biotin, chemiluminescent substances, enzymes, and the like, but are not particularly limited as long as they can be generally used for labeling a polymer substance. In addition, a protein substance or the like which easily binds to a labeling substance is previously bound to the hyaluronic acid-binding protein on the solid phase to which the high molecular weight hyaluronic acid is bound, and a A binding protein may be added together with the labeling substance. As described above, by further adding the hyaluronic acid-binding protein, the hyaluronic acid-binding protein immobilized on the solid phase, the high-molecular-weight hyaluronic acid, and the labeled or easily labelable hyaluronic acid-binding protein are added. Can be formed into a sandwich-like combination.

Next, the labeling substance of the sandwich-like conjugate is measured to quantify the high-molecular-weight hyaluronic acid. At this time, the sandwich-like conjugate sandwiches the high-molecular-weight hyaluronic acid, which is the object to be measured, while low-molecular-weight hyaluronic acid other than the object to be measured cannot form the above-mentioned sandwich-like conjugate. . Therefore, by measuring the concentration of the labeling substance on the surface of the solid phase, only the high molecular weight hyaluronic acid capable of forming the sandwich-like conjugate is determined. The method for measuring the concentration of the labeling substance differs depending on the labeling substance. For example, when a chemiluminescent substance is used as the labeling substance, the luminescence intensity of the solution after the reaction with the substance may be measured. In this case, for the labeling substance to be used and the high-molecular-weight hyaluronic acid that is the sample hyaluronic acid, prepare a sample of a known concentration, prepare a calibration curve for the relationship between the signal intensity and the known concentration, and calibrate the measured values. It is preferable that

The solid phase used in the above-described sandwich binding protein assay includes, but is not limited to, plates, tubes, beads, membranes, gels, and latexes. Any solid phase can be used as long as it can be fixed. The hyaluronic acid-binding protein used in the above-mentioned sandwich binding protein assay is described in A.I. E. FIG. Laurent (L
aurent) et al. (Analytical Biochemistry).
109, 386-394, 1980), link protein, hyaluronectin and the like.

The kit for measuring hyaluronic acid according to the present invention comprises:
For example, when the measurement method is a sandwich binding protein assay, basically, a solid phase to which a hyaluronic acid-binding protein is fixed, a buffer solution containing a surfactant, and a hyaluronic acid-binding protein for addition. And a labeling substance for the determination of sample hyaluronic acid.
The two components can be stored in separate containers, respectively, and stored as a kit that can be used according to the prescription when used. It is also possible to further add to the above-mentioned kit a block which covers a portion where the hyaluronic acid-binding protein is not fixed, a washing solution for the solid phase surface which is usually easily available, and the like. The hyaluronic acid-binding protein for addition may be labeled in advance with the labeling substance. Further, a portion of the surface of the solid phase to which the hyaluronic acid-binding protein is fixed, to which the hyaluronic acid-binding protein is not fixed, may be covered with a block. This block may be one selected from the group consisting of γ-globulin, serum albumin, and serum. The solid phase can be selected from the group consisting of plates, tubes, beads, membranes, gels, and latexes, and the labeling substance is selected from isotopes, fluorescent dyes, avidin, biotin, chemiluminescent substances, and enzymes. be able to.

In the above description, the method for measuring hyaluronic acid and the kit for measuring hyaluronic acid according to the present invention when the measuring method by the sandwich binding protein assay is used have been described.
The same can be applied to the case where other known measurement methods such as an aggregation method and a turbidimetric method are used.

[0017]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[0018]

Example 1 The effect of adding a surfactant according to the present invention was examined. [Materials and Methods] Materials: Buffer solution (0.1 M boric acid, 1% BSA, 2% polyethylene glycol) Reaction plate (hyaluronic acid binding protein coating) Conjugate solution (POD-labeled hyaluronic acid binding protein) Washing solution (PBS buffer solution) ) Coloring solution (TMB reagent) Reaction stopping solution (dilute sulfuric acid) Method: Sandwich binding protein assay 1) Add 100 μL of buffer solution to the reaction plate, add 10 μL of hyaluronic acid standard solution or serum sample, and stir.
It was left at room temperature for 60 minutes. 2) After washing the wells of the plate with the washing solution, 100 μL of the conjugate solution was added and left at room temperature for 30 minutes. 3) After washing the wells of the plate with the washing solution, the coloring solution 100
μL was added and left at room temperature for 30 minutes. 4) After adding 100 μL of the reaction stop solution and stirring, the absorbance at 450 nm was measured with an ultraviolet / absorptiometer. 5) A standard curve was prepared using the standard solution, and the concentration of hyaluronic acid in the serum sample was determined.

[Test Method] (1) Sodium chloride was added to a buffer solution at 0.5 M, 1.5 M,
And sodium chloride 0.5M and a polyoxyethylene alkyl phenyl ether as a surfactant (trade name: Emulgit, manufactured by Daiichi Kogyo Seiyaku; a mixture of polyoxyethylene alkyl phenyl ethers in which R is a nonyl group and n = 30 to 100 in the above chemical formula) 1% each, and a hyaluronic acid standard solution (0, 50, 100, 200, 60
(0,800 ng / mL) to prepare a standard curve for each buffer, and examined the effect of the buffer. (2) 0.5M, 1.5M, sodium chloride in buffer
2.0 M each was added, and the minimum amount of sodium chloride required for dissociation of hyaluronic acid in the serum sample was examined. (3) 0.15 M, 0.5 M, 1.0 M of sodium chloride was added to a buffer solution containing 1% of the surfactant, and the surfactant required for dissociation of hyaluronic acid in a serum sample was added. The effect of adding the agent was examined. (4) For the case where 1.5 M sodium chloride is added to the buffer, the case where only 0.5 M sodium chloride is added to the buffer and the case where 0.5 M sodium chloride and 1% of the surfactant are added to the buffer The correlation was compared and examined using serum samples.

[Results] 1. Investigation Results Using Hyaluronic Acid Standard Solution (FIG. 1) In general, surfactants are known as one of compounds that inhibit antigen-antibody binding reaction. Thus, the effect of the surfactant on the sandwich binding protein assay was confirmed using a standard solution prepared by adding sodium hyaluronate to a 1% BSA solution. Was not found (FIG. 1). In FIG. 1, OD 450 nm is 450 nm.
And Detergent means a surfactant (in this case, polyoxyethylene alkylphenyl ether). From these results, it was confirmed that the surfactant did not affect the binding reaction between hyaluronic acid and the binding protein. Furthermore, since there was no effect on the difference in the salt concentration of the buffer solution, it was confirmed that unlike the hyaluronic acid in the living body, the standard solution hyaluronic acid did not require a dissociation effect because it was in a free state. 2. Results of salt concentration study using serum samples (Figs. 2 and 3) The salt concentration in the buffer solution was examined using 10 serum samples. An equilibrium was reached (FIG. 2). In addition, 1
% The same study was conducted for the addition of the surfactant, and it was confirmed that the salt concentration became substantially equilibrium when the salt concentration was 0.5 M or more, and the salt concentration in the buffer solution was reduced to 1/3 due to the effect of the surfactant. (Figure 3). 3. Correlation study results using serum specimens (FIGS. 4 and 5) Using a buffer solution containing 1.5 M sodium chloride as a control, a buffer solution containing 0.5 M sodium chloride and 1%
Hyaluronic acid concentrations of 18 serum samples were measured using the buffer solution containing the surfactant and 0.5 M sodium chloride, and the correlation was determined. As a result, the slope was about 0.5 in the regression line in the case of 0.5 M sodium chloride, and it was confirmed that only about 50% of hyaluronic acid was measured (FIG. 4). In addition, in FIG. 4, HA means hyaluronic acid, x in y = 0.475x-10 is 1.5MNa
The concentration of HA (ng / mL) in Cl is 0.5 MN
represents the concentration of HA in aCl (ng / mL), respectively.
R represents a correlation coefficient between the two. On the other hand, in the case where the surfactant was added, the slope was 1.01 and it was confirmed that 100% was measured (FIG. 5). FIG.
In the formula, HA means hyaluronic acid, Detergent means a surfactant (in this case, polyoxyethylene alkylphenyl ether), and y = 1.014x-
X in 12 is the concentration of HA at 1.5 M NaCl (n
g / mL), and y is 0.5 MN with 1% surfactant.
represents the concentration of HA in aCl (ng / mL), respectively.
R represents a correlation coefficient between the two.

[Discussion] It has been confirmed that the surfactant dissociates proteins and the like bound to hyaluronic acid in serum and functions effectively in a hyaluronic acid measurement system. For this reason, by using a low salt concentration and the surfactant, it is possible to prevent crystal precipitation and a decrease in stirring efficiency. During the transition from the conventional method-based assay to the assay using an automatic analyzer, there was a concern about the obstacle due to the high salt concentration. However, the fact that the salt concentration can be reduced by using the surfactant is expected to make a great contribution.

[0022]

As described above, according to the measuring method of the present invention, it is possible to accurately measure hyaluronic acid at a lower ion concentration (salt concentration) than in the conventional method, and it is inevitable in the conventional method. There is no inconvenience such as a decrease in the stirring effect during the analysis due to precipitation of crystals, increased specific gravity, and increased viscosity. Therefore, the measurement method and the measurement kit of the present invention are effective for diagnosing inflammation such as rheumatism and liver fibrosis or for diagnosing cancer, and their technical value is extremely high.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of reactivity with and without the addition of a surfactant using a hyaluronic acid standard solution.

FIG. 2 is a graph showing the effect on measured values of hyaluronic acid in serum samples when no surfactant is added.

FIG. 3 is a graph showing the effect on the measured value of hyaluronic acid in a serum sample when a surfactant is added.

FIG. 4 shows the measurement of the hyaluronic acid concentration of 18 serum samples using a buffer solution containing 0.5 M sodium chloride as a control and a buffer solution containing 1.5 M sodium chloride when no surfactant is added. 4 is a graph showing the correlation.

FIG. 5 shows a buffer solution containing 1% surfactant (polyoxyethylene alkyl phenyl ether) + 0.5M sodium chloride as compared with a buffer solution containing 1.5M sodium chloride when no surfactant was added. FIG. 5 is a graph showing the correlation between the measured hyaluronic acid concentrations of 18 serum samples using FIG.

Claims (7)

[Claims] 1. A method for measuring hyaluronic acid, comprising using a surfactant in a measurement system. 2. The method according to claim 1, wherein the surfactant used in the measurement system is a nonionic surfactant. 3. The surfactant used in the measurement system is a polyoxyethylene alkyl phenyl ether represented by the following formula:
The method according to claim 1, wherein the method comprises at least one species. Embedded image (In the formula, R is a linear or branched alkyl group having 1 to 12 carbon atoms, and n is an integer of 30 to 100.) 4. The measuring method according to claim 1, wherein the measuring method is any one of a solid phase method, a competitive method, an agglutination method, and a turbidimetric method based on a binding protein assay method. 5. The method according to claim 1, wherein the sample is serum, plasma, synovial fluid, pleural effusion, tissue extract, culture supernatant, or urine. 6. A kit for measuring hyaluronic acid, comprising a surfactant and a reagent necessary for the measurement. 7. The measurement according to claim 6, wherein the reagent necessary for the measurement is a reagent used in any one of a solid phase method, a competitive method, an agglutination method, and a turbidimetric method based on a binding protein assay method. Kit.