JP2003265196A - Method for screening mucopolysaccharidosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method or the like for readily, efficiently and inexpensively screening mucopolysaccharidosis in high accuracy. <P>SOLUTION: The method for screening the mucopolysaccharidosis comprises the following steps (1) and (2): (1) a step for measuring absorbance at a specific wave length of a urine specimen obtained by bringing a solution obtained by reacting a glucosaminoglycan (GAG)-specific splitting enzyme with urine collected from a testee, into contact with a pigment changing the absorbance at the specific wave length by reacting with the GAG; and (2) a step for specifying the urine specimen having the amount of the GAG degraded by the GAG- specific splitting enzyme within a prescribed range by calculating the amount by using the measured result in the step (1). <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a screening method and a screening kit for mucopolysaccharidosis.

[0002]

2. Description of the Related Art A technique or the like closest to the present invention will be described below.

Clinica Chimica Acta, vol.264, p245-25
0 (1997), urinary glycosaminoglycan (hereinafter referred to as G
AG) is measured using 1,9-dimethylmethylene blue (hereinafter, dimethylmethylene blue is referred to as “DMMB”) to screen for mucopolysaccharidosis. In addition, it is described that this dye also reacts with an acrylic acid polymer contained in a general paper diaper to give a false positive result.

In addition, Chimica et Biophysica Acta, vol.
883, p173-177 (1986) describes that the absorption wavelength of 1,9-DMMB is 595 nm when not reacting with GAG, whereas it changes to 525 nm when reacting with GAG.

Further, Japanese Patent Laid-Open No. 1354396/1992 discloses that
Decomposing GAG into a disaccharide by a GAG-specific degrading enzyme,
The composition of the disaccharide was analyzed by high performance liquid chromatography (hereinafter,
The method of analyzing GAG by analyzing it by HPLC) is described.

However, none of these documents describes or suggests the screening method and kit of the present invention.

[0007] Mucopolysaccharidoses is
It is a general term for a series of genetic diseases caused by disorders (reduction of activity) of enzymes related to the degradation and metabolism of GAG. It is known that specific GAGs are accumulated in tissues and secreted in urine depending on the type of enzyme to be damaged. The clinical manifestations of mucopolysaccharidoses are variable, but most are associated with rough facial features, multiple bone abnormalities, and visceral enlargement. It may also be accompanied by hearing loss, cardiovascular disorders and delayed mental development.

[0008] Mucopolysaccharidoses usually have no symptoms at birth, and are manifested by symptoms such as short development of height in infants and children, abnormal growth of bones, and hair depth. Also, at birth, there may be a gradual delay in mental development over the years. Therefore, if mucopolysaccharidosis is diagnosed early after birth, when symptoms do not yet appear, enzyme replacement therapy or gene therapy can be performed early,
It may be possible to stop delays in mental development. Therefore, it is most desirable that all neonates be diagnosed with mucopolysaccharidosis.

[0009]

However, while the number of newborns in Japan exceeds 1 million per year, the incidence of mucopolysaccharidosis is extremely low at 1 in 40,000 to 50,000, and the diagnosis is enzyme deficiency or enzyme. Since it is complicated and expensive to investigate an abnormality, a highly accurate screening method is required as a pre-stage of the diagnosis. In particular, it is difficult to directly collect the necessary amount of newborn urine when needed, so it is easy to extract it from a paper diaper, but general-purpose pigments react not only with GAG but also with the components of the paper diaper. It is necessary to eliminate such factors as they give false positives.

By solving these problems, patients with mucopolysaccharidosis are not leaked (without leaking false-negative patients), accurately (prevent false-positive patients as much as possible), simple, and
If a method that can be efficiently and inexpensively screened can be provided, it becomes possible to detect the possibility of mucopolysaccharidosis in all newborns, and it is possible to detect mucopolysaccharidosis patients without omission.
It becomes possible to treat early after an accurate definite diagnosis.

Not only that, the frequency of false-positive patients undergoing unnecessary diagnosis is reduced, and the burden on the medical field is also reduced. Therefore, high-quality definitive diagnosis and treatment can be expected accordingly. That is, the present invention is highly accurate for mucopolysaccharidosis,
It is an object to provide a convenient, efficient and inexpensive screening method and kit.

[0012]

Means for Solving the Problems As a result of intensive investigations by the present inventors to solve the above problems, as a result of providing a step of appropriately combining a GAG-specific degrading enzyme and a general-purpose dye, high precision and simple They have found that efficient and inexpensive screening for mucopolysaccharidosis is possible, and completed the present invention.

That is, the present invention provides a method for screening mucopolysaccharidosis (hereinafter, referred to as "method 1 of the present invention"), which comprises at least the following steps (1) and (2). (1) Obtained by contacting "a solution obtained by causing an enzyme that specifically decomposes GAG to act on urine collected from a subject" and "a pigment whose absorbance at a specific wavelength changes in response to GAG" Measuring the absorbance of the urine sample at the specific wavelength. (2) Using the result measured in step (1), GA
A step of calculating a GAG amount decomposed by an enzyme that specifically decomposes G, and identifying a urine sample within the specified range.

The "urine collected from the subject" in the method 1 of the present invention is preferably collected from the subject corresponding to the urine sample identified through the step including at least the following steps (A) and (B). (A) A step of measuring the absorbance at a specific wavelength of a urine sample obtained by contacting the urine of a subject with “a dye whose absorbance at a specific wavelength changes in response to GAG”. (B) Using the results measured in (A), calculate the amount of the substance that has reacted with the “dye that changes the absorbance at a specific wavelength in response to GAG” and identify the urine sample that is outside the reference range Step. The present invention also includes the following step (3)
There is provided a screening method for mucopolysaccharidosis (hereinafter, referred to as “method 2 of the present invention”) containing at least: (3) A step of analyzing a GAG composition in urine collected from a subject corresponding to the urine sample identified by the step (2) described in the method 1 of the present invention.

The present invention also provides a method for screening mucopolysaccharidosis which comprises at least the following step (4):
"Invention Method 3") is provided. (4) A step of associating the GAG composition analyzed by the step (3) of the method 2 of the present invention with mucopolysaccharidosis.

The dye whose absorbance at a specific wavelength changes in response to GAG used in the method of the present invention is DM
It is preferably MB. Similarly, the “enzyme that specifically decomposes GAG” is preferably one or more enzymes selected from chondroitinase, keratanase and heparitinase. Further, GA in the method 2 of the present invention
The G composition is preferably analyzed by causing an enzyme that specifically decomposes GAG to act on it to decompose GAG into disaccharides and analyzing the composition of the disaccharides. Note that, hereinafter, when simply referred to as “the method of the present invention”, the methods 1 to 3 of the present invention
Should be understood as a concept that refers to either of.

The present invention also provides a mucopolysaccharidosis screening kit (hereinafter referred to as "the kit of the present invention") containing at least the following components (a) and (b). (A) a dye whose absorbance at a specific wavelength changes in response to GAG (b) an enzyme which specifically decomposes GAG "a dye whose absorbance at a specific wavelength changes in response to GAG" is It is preferably DMMB. Similarly, "enzyme that specifically decomposes GAG"
Is preferably one or more enzymes selected from chondroitinase, keratanase and heparitinase.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. <1> Method 1 of the Present Invention Method 1 of the present invention is a screening method for mucopolysaccharidosis, which comprises at least the following steps (1) and (2). (1) Obtained by contacting "a solution obtained by causing an enzyme that specifically decomposes GAG to act on urine collected from a subject" and "a pigment whose absorbance at a specific wavelength changes in response to GAG" Measuring the absorbance of the urine sample at the specific wavelength. (2) Using the result measured in step (1), GA
A step of calculating a GAG amount decomposed by an enzyme that specifically decomposes G, and identifying a urine sample within the specified range.

Here, the "urine collected from the subject" is preferably collected from the subject corresponding to the urine sample identified through the step including at least the following steps (A) and (B). (A) A step of measuring the absorbance at a specific wavelength of a urine sample obtained by contacting the urine of a subject with “a dye whose absorbance at a specific wavelength changes in response to GAG”. (B) Using the results measured in (A), calculate the amount of the substance that has reacted with the “dye that changes the absorbance at a specific wavelength in response to GAG” and identify the urine sample that is outside the reference range Step.

The "step including at least the above steps (A) and (B)" which is preferably carried out in the preceding step of the method 1 of the present invention is carried out by collecting urine from a very large number of subjects, who may have mucopolysaccharidosis. This is a screening step that is simple, efficient, and inexpensive without omission, and can be called a so-called primary screening.

The method of collecting urine in step (A) and step (1) described later is not particularly limited.
For example, it may be directly collected when the subject urinates, or may be extracted from a diaper (any material may be used as long as it does not contain GAG). Since the method of the present invention is preferably used in a newborn baby in which urination control is particularly difficult,
It is extremely simple in that urine can be collected from diapers and the like.

The urine subjected to the above step (A) may be freshly collected, or may be stored as long as it does not affect the screening result obtained by the method of the present invention. Good. For example, insolubilization and decomposition of GAG in urine that occurs during storage will affect screening results, but preservatives such as sodium azide will prevent such effects, and surfactants such as Tween 20 and guanidine hydrochloride The effect can be eliminated by the denaturing agent.

Further, ethylenediaminetetraacetic acid (EDT
A), zinc ion, cobalt ion and the like affect the screening result by inhibiting the action of the enzyme that specifically decomposes GAG in the above step (1), and thus stabilization containing such a substance is performed. It is preferable not to add an agent or the like.

Further, since a substance having an absorption peak close to that of the dye used in the step (A) also affects the screening result, it is preferable not to add such a substance.

The "dye whose absorbance at a specific wavelength changes in response to GAG" used in the above step (A) is G
A dye whose absorption at a specific wavelength of the dye changes to a detectable level by reacting with AG, and
There is no particular limitation as long as it is soluble in urine. Here, “change in absorbance” is a concept including both “increased absorbance” and “decreased absorbance”, but “increased absorbance” is preferable. Further, the “detectable change” may be any change that can be detected by an analytical instrument or the like.

However, it is preferable to avoid a dye near the wavelength of the absorption peak of the dye contained in urine itself because it is easily affected by the dye in urine. Examples of dyes that can be used here include Alcian blue, Stainsall,
Examples include cupro meronic blue. In addition, "G
The "dye whose absorbance changes at a specific wavelength in response to AG" includes not only a dye whose absorbance changes at one wavelength by reacting with GAG but also a dye whose absorbance changes at a plurality of wavelengths. It

Examples of the latter dyes include GA
Dyes that react with G to increase the absorbance at a certain wavelength and decrease the absorbance at another wavelength are exemplified and preferred. Among them, GA
Dyes that react with G to increase the “absorbance at the wavelength of the absorption peak exhibited by the dye reacted with GAG” and decrease the “absorbance at the wavelength of the absorption peak exhibited by the unreacted dye” are preferred. When such a dye is used, after contact with the dye, one of "absorbance at wavelength of absorption peak exhibited by the dye reacted with GAG" and "absorbance at wavelength of absorption peak exhibited by unreacted dye" It is possible to measure only one or both. It is preferable to measure both of them, because the reaction with GAG can be detected with higher sensitivity.

When such dyes are used, those having extremely small difference between one "wavelength" and another "wavelength" (for example, change in absorption peak wavelength before and after reaction with GAG) are avoided. It is preferable. Specifically, the difference between the two wavelengths (for example, the change in the wavelength of the absorption peak before and after the reaction) is 30
It is preferable to use a dye having a wavelength of not less than 50 nm,
It is more preferable to use a dye having a thickness of at least nm.
DMMB etc. are illustrated as such a pigment | dye. In particular
1,9-DMMB is also preferred because it is widely used and inexpensive.

In such a dye, a certain wavelength (for example, the wavelength of the absorption peak exhibited by the dye reacting with GAG) and another wavelength (for example, the wavelength of the absorption peak exhibited by the unreacted dye concerned) are transmitted to GAG. It varies depending on the type of dye that reacts to change the absorbance at a specific wavelength, and its value is already known or can be appropriately determined by those skilled in the art.

For example, when the dye is 1,9-DMMB,
The "wavelength of the absorption peak exhibited by the dye reacting with GAG" is in the range of 520 to 530 nm, and the "wavelength of the absorption peak exhibited by the unreacted dye is 585-595n."
The range is m.

When such a dye is used, the “absorbance at the wavelength of the absorption peak exhibited by the dye reacting with GAG” is increased by the reaction with GAG, while the “wavelength of the absorption peak exhibited by the unreacted dye is increased. Sensitivity results are obtained because the "absorbance at" decreases.

In the step (A), the method of "contacting" the urine with the dye is such that the component in the urine and the dye molecule can be brought into contact with each other, and the dye molecule after the contact is dissolved in the urine. The method is not particularly limited as long as it is a method. For example, urine or solution may be contacted by adding a dye, urine or solution may be added to contact the dye, or both may be simultaneously added for contact.

Alternatively, a method of contacting urine with a filter paper or the like on which a dye is adsorbed and checking the color tone of the filter paper or the like may be adopted. The temperature at the time of contact between the solution and the dye is 4 ° C to 40 ° C.
The temperature may be about 30 ° C, preferably about 15 ° C to 30 ° C,
About 25 ° C. is particularly preferable. In general, the change in the absorbance of the dye reacted with GAG occurs rapidly, but since it gradually changes, the absorbance is changed within 30 minutes, preferably within 10 minutes, and most preferably within 5 minutes after the contact with the dye. It is desirable to measure. The method for measuring the absorbance at a specific wavelength is not particularly limited, and it can be measured using an ordinary absorptiometer or the like.

In the step (B), the amount of the substance reacted with the "dye whose absorbance at a specific wavelength changes in response to GAG" (hereinafter simply referred to as "reaction with dye") is used by using the result measured in (A). (Also referred to as the amount of substance that has been processed)), and this is the step of identifying a urine sample outside the reference range.
The method of calculating the “amount of substance that has reacted with the dye” using the result (measured value of absorbance) measured in (A) is not particularly limited. The “amount of substance reacted with the dye” may be calculated as “value of concentration” or “value of absorbance”. When a dye that increases the absorbance at a certain wavelength by reacting with GAG and decreases the absorbance at another wavelength is used as the dye,
The “amount of substance reacted with the dye” may be calculated as “a value of a ratio of the absorbance at a certain wavelength to the absorbance at another wavelength”.

For example, when the "amount of substance reacted with the dye" is calculated as the "concentration value", the "absorbance value" (or a certain value) after contacting the dye with a GAG standard solution having a known concentration is used. A calibration curve or relational expression is created in advance for the relationship between the GAG concentration and the value of the ratio of the absorbance at a wavelength to the absorbance at other wavelengths, and the measurement result for the sample of unknown concentration is obtained by the calibration curve or the relational expression. It can be obtained by converting to GAG concentration using the above. Further, the concentration may be corrected based on the concentrations of other components such as creatinine contained in the urine sample, which is preferable.

The "reference range" in the above step (B) means the amount of substance (in urine) that reacts with "a dye whose absorbance at a specific wavelength changes in response to GAG".
Means a range in which is determined to be healthy, and serves as a criterion for deciding whether or not to proceed to the next screening step.

It is possible to say that urine specimens having the “amount of substance reacting with pigment” calculated as described above outside this reference range (healthy range) may have mucopolysaccharidosis. Need to be identified. The urine sample specified here needs to proceed to the next step (step (1)).

The "reference range" can be set as a range in which the "amount of substance reacting with the pigment" is obtained in advance for a plurality (as many as possible) of urine samples of healthy persons and the value is distributed. As a guide, an amount of 0 to 20 μg / mg Cre (Cre means creatinine) is exemplified.

The "step including at least the above steps (A) and (B)" which is preferably performed in the preceding stage of the method 1 of the present invention may further include other steps. For example, before the step (A), a step of pretreating the urine of the subject using a protease, a protein precipitating agent, a desalting column or the like may be added.

For example, when "a dye whose absorbance at a specific wavelength changes in response to GAG" is used, the above steps (A) and (B) can be performed as follows. In the parentheses, the case where 1,9-DMMB is used as the dye is shown.

Step (A): The urine of the subject and "GAG
A dye that changes its absorbance at a specific wavelength in response to
Regarding the urine sample obtained by contacting with (1,9-DMMB), "wavelength of absorption peak exhibited by the dye reacting with GAG" (530 nm) and "wavelength of absorption peak exhibited by the unreacted dye" ( 590 nm).

Step (B): "Absorbance at wavelength of absorption peak exhibited by the dye reacting with GAG by reaction of GAG with the dye" (530 nm) using the result measured in step (A) And "Absorbance at wavelength of absorption peak of unreacted dye"
The ratio of both (590 nm) is calculated. As a result, the increased amount of the former and the decreased amount of the latter due to the reaction with the dye can be detected with high sensitivity, and the urine sample that may contain GAG can be screened without omission. The method for calculating the ratio is not particularly limited, but "absorbance at a wavelength of an absorption peak exhibited by the dye reacting with GAG by reacting GAG with the dye" / "at a wavelength of an absorption peak exhibited by the unreacted dye" Assuming "absorbance" (530 nm / 590 nm), the larger this value, the more substances that react with the dye are contained in the urine sample. If the numerator and the denominator are reversed, (590nm / 530n
m), the smaller this value, the more substances that react with the pigment are contained in the urine sample. The value of such a ratio (the value of the ratio between the absorbance at a certain wavelength and the absorbance at another wavelength) may be used as it is as the “amount of the substance reacted with the dye”. Alternatively, using a GAG standard solution having a known concentration, the “absorbance at a certain wavelength (530 n
m) and the value of the ratio of the absorbance (590 nm) at other wavelengths "to the GAG concentration, using the calibration curve and the relational expression prepared in advance, the amount of the substance reacted with the dye May be calculated as Further, the concentration may be corrected based on the concentrations of other components such as creatinine contained in the urine sample, which is preferable.

In step (1), "a solution obtained by reacting urine collected from a subject with an enzyme that specifically decomposes GAG" and "a dye whose absorbance changes at a specific wavelength in response to GAG" Is a step of measuring the absorbance at the specific wavelength of the urine sample obtained by contacting with. The "urine collected from the subject" is preferably collected from the subject corresponding to the urine sample identified through the step including at least the steps (A) and (B).

In step (A), the "urine of the subject" is brought into contact with the dye, but in step (1), the "solution obtained by causing the enzyme that specifically decomposes GAG to act on the urine collected from the subject" is added. They differ in that they come into contact with the dye.

The "enzyme that specifically decomposes GAG" (hereinafter referred to as "GAG-specific decomposing enzyme") has a reactivity to "a dye whose absorbance at a specific wavelength changes in response to GAG" by acting on GAG. There is no particular limitation as long as it is an enzyme that decomposes GAG to such an extent that it disappears. As such an enzyme, one or more enzymes selected from chondroitinase, keratanase and heparitinase are exemplified and preferred. Specifically, the following is exemplified by the type of GAG to be decomposed.

Chondroitin sulfate (hereinafter referred to as CS): chondroitinase ABC, chondroitinase ACII, chondroitinase ACI dermatan sulfate (hereinafter referred to as DS): chondroitinase ABC, chondroitinase B keratan sulfate (hereinafter referred to as KS) ): Keratanase, Keratanase II, Heparan sulfate (hereinafter referred to as HS): Heparitinase
I, heparitinase II, and these GAs for complete degradation of GAG in urine.
It is preferable to use a G-specific degrading enzyme in combination.

The method for causing the GAG-specific degrading enzyme to act is not particularly limited as long as the GAG molecule in urine and the enzyme molecule come into contact with each other to cause an enzymatic reaction. For example, the enzyme solution may be contacted with urine in the solution, or the immobilized enzyme having the enzyme bound to an appropriate solid phase (beads, membrane, etc.) may be contacted with urine. The former is preferred.

Those skilled in the art can appropriately determine the conditions under which the GAG-specific degrading enzyme acts, depending on the enzyme used. For example, the optimum temperature and optimum pH of the following enzymes are generally as follows.

Chondroitinase ABC: optimum temperature 3
7 ° C, optimum pH 8.0 chondroitinase ACII: optimum temperature 37 ° C, optimum p
H 6.0 Keratanase II: optimum temperature 37 ° C, optimum pH 6.0 heparitinase I: optimum temperature 37 ° C, optimum pH 7.0 heparitinase II: optimum temperature 37 ° C, optimum pH 7.0 When using enzymes together and reacting at the same time, pH
Under the conditions of about 5.5 to 8.0 and a temperature of 25 to 37 ° C, 1
It is preferable to react for about 2 hours.

In the "urine collected from the subject", the above "G
When "AG-specific degrading enzyme" is allowed to act under the above-mentioned method and conditions, when GAG is contained in the urine, the G
AG is decomposed by an enzyme. If urine contains a substance other than GAG that reacts nonspecifically with "a dye whose absorbance at a specific wavelength changes in response to GAG", the substance is It remains in urine without being decomposed by enzymes.

Then, when the solution after the enzymatic reaction is brought into contact with "a dye that reacts with GAG to change the absorbance at a specific wavelength" and reacted, the sample containing the GAG in urine is treated with the GAG by the enzyme. Since it is decomposed, it does not react with the dye and the absorbance does not change. On the other hand, in the urine, G
A sample containing a substance other than AG that reacts nonspecifically with a "dye whose absorbance at a specific wavelength changes in response to GAG" remains in the sample without being decomposed by the enzyme. Therefore, the absorbance changes in response to "a dye whose absorbance at a specific wavelength changes in response to GAG".

The step (2) is a step of calculating the GAG amount decomposed by the GAG-specific degrading enzyme using the result measured in the step (1) and specifying the urine sample within the specified range. is there.

The "GAG amount" referred to here is the "concentration value".
Or may be calculated as “value of absorbance”. When a dye that reacts with GAG to increase the absorbance at a certain wavelength and decreases the absorbance at another wavelength is used, the "GAG amount" is defined as "the absorbance at a certain wavelength and the other wavelength." The value of the ratio with the absorbance at.
The calculation of the “GAG amount” including this point is the same as the description regarding the calculation of the “amount of the substance reacted with the dye” in step (B), unless otherwise noted.

In the step (1), when the solution after the enzymatic reaction is brought into contact with "a dye whose absorbance at a specific wavelength changes in response to GAG" and reacted, the urine before the enzymatic reaction contains a substance other than GAG. Therefore, when a “substance that reacts nonspecifically with the dye” (nonspecific reaction substance) is contained, the absorbance of the dye changes depending on the amount of the nonspecific reaction substance. The absorbance of the dye does not change when only the dye is included.

Therefore, when the solution after the enzymatic reaction is brought into contact with "a dye whose absorbance at a specific wavelength changes in response to GAG", the change in the absorbance depends on the substance that reacts nonspecifically with the dye. It will reflect the quantity.

Urine contains both GAG and a substance other than GAG that reacts nonspecifically with "a dye whose absorbance at a specific wavelength changes in response to GAG". There is also a possibility. Therefore, in step (1), a "sample that causes a GAG-specific degrading enzyme to act" and a "sample that does not act on the enzyme" are provided,
In the step (2), the "GAG amount" is obtained by obtaining the difference or ratio between the value in the sample without the GAG-specific degrading enzyme and the value in the sample with the GAG-specific degrading enzyme.
It is desirable to calculate

The "difference" between the value in the sample in which the GAG-specific degrading enzyme does not act and the value in the sample in which the GAG-specific degrading enzyme acts is directly indicated as "GAG amount". Therefore, the large absolute value of this difference means that the GAG amount is large, and the substantial absence of this difference means that GAG does not substantially exist (“at a specific wavelength in response to GAG, It means that only a substance that reacts nonspecifically is included in the “dye whose absorbance changes”).

Also, the ratio of these (for example, "GAG
Value in sample treated with specific degrading enzyme ”/
The "value in a sample that does not act on a GAG-specific degrading enzyme" means the content ratio of the amount of a substance that reacts nonspecifically with "a dye whose absorbance at a specific wavelength changes in response to GAG". . Therefore, this ratio is 1
The fact that it is close to 00% means that the content ratio of the substance is high (the content ratio of GAG is close to 0),
When this ratio is close to 0%, it means that the content ratio of the substance is close to 0 (containing GAG). Therefore, if this ratio is X%, 100-X
(%) Indicates "GAG amount" (content ratio of GAG).

By the evaluation as described above, the influence (false positive) due to a substance other than GAG which reacts nonspecifically with "a dye whose absorbance at a specific wavelength changes in response to GAG" (false positive) It can be eliminated and the GAG amount can be accurately measured.

The “specified range” in step (2) means the range of the amount of GAG in urine that is suspected to be a possibility of mucopolysaccharidosis, and whether or not to proceed to the next screening step. It is a standard for determining whether or not.

As described above, in the case of a urine sample in which the calculated “GAG amount” is within the specified range, it is possible that the urine contains a sufficient amount of GAG that is suspected to be mucopolysaccharidosis. It is necessary to identify this because it is highly likely. Then, by identifying the urine sample within the “specified range”, the urine sample outside the “specified range” can be excluded. That is, a urine sample (a urine sample showing a false positive) containing a substance that reacts nonspecifically with “a dye whose absorbance at a specific wavelength changes in response to GAG” is excluded. In this sense, it can be said that the “specified range” is a range for specifying a true positive case.

The "specified range" can be set as a range in which the GAG amount is obtained in advance for a plurality (as many as possible) of urine samples of mucopolysaccharidosis patients and the value is distributed. When the amount of GAG (content ratio of GAG) is shown as "the ratio of the measured value in the sample to which the GAG-specific degrading enzyme is allowed to act and the measured value in the sample not allowed to act", the standard for the "specified range" is For example, about 40 to 100% is exemplified.

The method 1 of the present invention, that is, the “screening method for mucopolysaccharidosis comprising at least the above steps (1) and (2)”, may further include other steps. For example, before the step (1), a step including at least the steps (A) and (B) or a step of pretreating the urine of the subject may be added.

It is necessary to proceed to the next step (step (3) in the method 2 of the present invention) because it can be said that the sample having the measured value in the step (2) within the specified range may have mucopolysaccharidosis. There is.

<2> Method 2 of the Present Invention Method 2 of the present invention is a screening method for mucopolysaccharidosis, which comprises at least the following step (3). (3) GAG in urine collected from a subject corresponding to the urine sample identified in step (2) of method 1 of the present invention
Step of analyzing the composition.

The method for analyzing the GAG composition in urine is not particularly limited as long as it can analyze the type of GAG contained in urine. Examples of the method for analyzing the GAG composition in urine include the following. i) Degrading enzyme specific to a specific type of GAG in urine
And a difference in elution time of a plurality of decomposition products (disaccharides) produced by the action on the ion exchange column is analyzed by high performance liquid chromatography (HPLC) (disaccharide analysis). ii) A method of analyzing the difference in mobility depending on the type (charge) of GAG using electrophoresis. iii) A method of analyzing by ELISA using an antibody that specifically binds to a specific type of GAG. iv) urinary GA containing a specific type of GAG-specific degrading enzyme
GAG is used to determine the presence or absence of GAG decomposition and the degree of GAG decomposition.
A method of analysis using a dye that reacts with.

Among these, disaccharide analysis is preferable from the viewpoint that a trace amount of GAG can be analyzed with high sensitivity and high accuracy. That is, G is caused by the action of a GAG-specific degrading enzyme.
It is preferably performed by decomposing AG into a disaccharide and analyzing the composition of the disaccharide. Disaccharide analysis is described in JP-A-4-
It can be performed by the method described in Japanese Patent No. 135496.

<3> Method 3 of the Present Invention Method 3 of the present invention is a screening method for mucopolysaccharidosis, which comprises at least the following step (4). (4) A step of associating the GAG composition analyzed by the step (3) of the method 2 of the present invention with mucopolysaccharidosis.

It is known that the relationship between the types of mucopolysaccharidosis and the corresponding GAG excreted in urine is as shown in Table 1 below.

[0070]

[Table 1]

The association between the GAG composition analyzed by step (3) of the method 2 of the present invention and mucopolysaccharidosis can be performed according to this table.

First, if the analyzed GAG composition is a composition containing GAG as described above, it can be associated with some kind of mucopolysaccharidosis or with a high possibility.

Then, for example, the analyzed GAG composition is
If the composition contains DS and HS, I
It can be associated with, or is likely to have, type II, type II or type VII mucopolysaccharidosis. Further, for example, when the analyzed GAG composition is a composition containing only HS, it can be associated with the type III mucopolysaccharidosis or the possibility thereof. Similarly, the following can be related based on Table 1.

The methods 1 to 3 of the present invention can be carried out by different persons. For example, the method 1 of the present invention is carried out at a medical site (hospital, clinic, etc.), the method 2 of the present invention is carried out by a trader (inspection center, etc.) based on the result, and the method 3 of the present invention is further carried out based on the result. It is also possible to carry out by another company. Of course, the method 1 of the present invention
It is also possible for one person to carry out all of steps 3 to 3. Also,
Inventive methods 1 and 2 are carried out in a medical field, and invented method 3
It is also possible that the method is carried out by a trader, and similarly, the method 1 of the present invention is carried out at a medical site, and the methods 2 and 3 of the present invention can also be carried out by a trader.

<4> Kit of the Present Invention The kit of the present invention is a screening kit for mucopolysaccharidosis containing at least the following components (a) and (b). (A) Dye whose absorbance at a specific wavelength changes in response to GAG (b) GAG-specific degrading enzyme

The kit of the present invention is not particularly limited as long as it contains at least the above (a) and (b), and further,
Diluent for diluting urine, urine pretreatment liquid, preservative, stabilizing liquid, anion exchanger for disaccharide analysis, disaccharide standard substance for disaccharide analysis, known standard for preparing calibration curve Glycosaminoglycan standards of concentration, microtiter plates, etc. can be added as constituents.

As the dye whose absorbance at a specific wavelength changes in response to GAG, DMMB is used as in the method of the present invention.
Is preferred. The GAG-specific degrading enzyme is also preferably one or more enzymes selected from chondroitinase, keratanase and heparitinase, as in the method of the present invention.

These constituent components can be housed in separate containers and stored as a kit which can be used in accordance with the measuring method of the present invention at the time of use. The screening for mucopolysaccharidoses using the kit of the present invention is performed according to the above <1>.
~ <3> The method of the present invention can be used.

[0079]

EXAMPLES The present invention will now be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 Regarding steps (A) and (B), which are the pre-stages of the method 1 (steps (1) and (2)) of the present invention, human urine was used to determine "absorbance at a specific wavelength in response to GAG. The following experiment was conducted to examine whether or not screening with "dye that changes in color" is possible.

(1) Preparation of calibration curve Various GAGs (HS (derived from pig lung), CS-A (derived from bovine trachea), CS-C (derived from shark cartilage) or DS (derived from pig lung);
All are made by Seikagaku Corporation, standard solution (concentration: 1,
To 2, 3, 4, 6, 8, 12, and 16 μg / mL), add 50 μL of reaction buffer (2.88M guanidine hydrochloride-0.05% Tween20-50 mM sodium acetate buffer), and then add DMM.
400 μL of B dye solution (15 mg / L DMMB -75 mM sodium formate buffer (pH 6.8)) was added and stirred, and the wavelength was 530 nm.
The absorbance at 590 nm and 590 nm was measured with an absorptiometer to determine the ratio between them (OD530nm / OD590nm). GAG concentration and OD53
The relationship (calibration curve) with 0 nm / OD590 nm is shown in FIG. In addition,
Circles in FIG. 1 indicate HS, triangles indicate CS-A, diamonds indicate CS-C, and squares indicate DS.

As a result, it was shown that the reactivity of HS is slightly lower than that of CS and DS, but the reactivity of DMMB does not differ greatly depending on the type of GAG.

(2) Measurement of GAG in urine GAG in urine was measured by the following method using urine collected from 16 patients suspected of having some disease and 9 healthy subjects.

1) Add physiological saline to 100 μL of urine and dilute it 4 times. 2) Reaction buffer (2.88M guanidine hydrochloride-0.075% Tw
een20-150 mM sodium acetate buffer (pH 6.8)) 50 μL
Is added to the microtube. 3) Add the urine sample prepared in 1) to the microtube prepared in 2). 4) DMMB dye solution (15mg / L) in the urine sample prepared in 3)
DMMB-75 mM sodium formate buffer (pH 6.8)) 400
Add μL and stir. 5) Measure the absorbance at wavelengths of 530 nm and 590 nm with an absorptiometer and obtain the ratio (OD530nm / OD590nm) of both. 6) Using the calibration curve created in (1) above, GA in each sample
Calculate the G concentration.

The measurement results are shown in Table 2. In addition, Table 3 shows the results of measurement using the same urine sample again on the same day by the same method. In addition, "nd" in the table indicates that it was not detected. "MSD" means multiple sulfatase deficiency.

[0085]

[Table 2]

[0086]

[Table 3]

As is clear from Tables 2 and 3, these 2
It can be said that this method has a very high reproducibility because the measured values of GAG match well between the two measurements. Moreover, the amount of GAG in urine of healthy subjects was about 9 to 16 μg / mg Cre (mean: 12 to 13 μg / mg Cre), whereas all mucopolysaccharidic patients showed much higher values.
From the above results, it was shown that mucopolysaccharidosis can be selected without leakage and at low cost by selecting a urine sample that falls outside the range (reference range) of urinary GAG amount of healthy subjects.

[0088]

Example 2 Regarding the method 1 (steps (1) and (2)) of the present invention, human urine was used, and a GAG-specific degrading enzyme and a dye whose absorbance at a specific wavelength was changed in response to GAG were used. The following experiment was conducted to examine whether screening is possible. (1) GAG addition test First, GAG was measured using a sample obtained by adding GAG to urine of a healthy person. The added GAGs are as follows. HS (derived from Bota aorta), CS-C (derived from shark cartilage)
Or KS (derived from bovine cornea) (all manufactured by Seikagaku Corporation).

The GAG-specific degrading enzymes (all manufactured by Seikagaku Corporation) were used in the following amounts per 100 μL of urine. 2.5 mU of heparitinase for HS
Mixed solution of I and II (100 μL). 80 mU chondroitinase ABC and 16 mU chondroitinase ACII (100 μL) for CS. 1 mU of keratanase for KS
II (100 μL).

The enzyme treatment and GAG measurement were carried out as follows. 1) To 90 μL of urine, add 10 μL of a 200 μg / ml solution of any of the above GAGs or 0.9% NaCl. 2) To the solution prepared in 1), 100 μL of the above-mentioned amount of GAG-specific degrading enzyme or 100 mM sodium acetate buffer (pH 6.0)
Added. 3) Incubate at 37 ℃ for 2 hours. 4) Add 200 μl of 0.9% NaCl and stir. 5) Measure with DMMB. Specifically, it is as follows. 5-1) Dispense the solution prepared in 4) above and the GAG standard solution (prepared with the corresponding GAG) into a microtube. 5-2) Add 50 μL of reaction buffer to this. 5-3) Add 400 μL of DMMB dye solution and stir. 5-4) 300 μL of the solution prepared in 5-3) is transferred to the well of a 96-well microtiter plate (trade name: Maxisorp, manufactured by NUNC). 5-5) In the same manner as above, the absorbances at wavelengths of 530 nm and 590 nm are measured, and the ratio between them (OD530nm / OD590nm) is determined.

As a result of carrying out this test, in the case where heparitinase I and II were allowed to act on HS-added urine, the measured value of added HS (OD530nm / OD590nm) was 63 to 98.
% Was reduced by enzyme treatment. In addition, in the case where chondroitinase ABC and chondroitinase ACII were allowed to act on CS-C-added urine, 99 to 100% of the added CS-C measured value (OD530nm / OD590nm) was reduced by the enzyme treatment. . In addition, in the case where keratase II was allowed to act on urine to which KS was added, 63 to 100% of the measured value (OD530nm / OD590nm) of added KS was decreased by the enzyme treatment.

From this result, the increase in the measured value (OD530nm / OD590nm) accompanying the addition of GAG was decreased by at least 63% or more by each degrading enzyme (the ratio (residual ratio) after the enzyme treatment to that before the enzyme treatment was Less than 37%). From this result, in the case where an increase in the measurement value due to the reaction with the dye was observed in the above steps (A) and (B), if it was due to the increase in urinary GAG, this step ( It was revealed that the measured value in 1) was lower than the measured value in step (A).

(2) Test using an actual sample GAG was measured using urine collected from a total of 9 mucopolysaccharidosis patients. The following GAG-specific degrading enzyme mixed solution was used for 100 μL of urine.

A mixture of 2.5 mU of heparitinase I and II, 80 mU of chondroitinase ABC, 16 mU of chondroitinase ACII and 1 mU of keratanase II, 100 mM sodium acetate buffer (pH 6.0) in total volume. 100μ
A mixed solution designated as L.

The enzyme treatment and GAG measurement were performed as follows. 1) To 90 μL of urine, 300 μL of the above GAG-specific degrading enzyme mixed solution or 100 mM sodium acetate buffer (pH 6.0) is added. 2) Incubate at 37 ℃ for 2 hours. 3) Add 100 μL of 0.9% NaCl and stir. 4) Measure with DMMB. Specifically, it is the same as the above (1). The results of this test are shown in Table 4 together with the residual rate (ratio of the measured value before the enzyme treatment to the measured value before the enzyme treatment).

[0096]

[Table 4]

From Table 4, it can be said that at least the amount corresponding to the difference between the measured values before and after the enzyme treatment corresponds to GAG. From these results, in the method 1 of the present invention (steps (1) and (2)), GAG present in urine
Other substances other than those that react non-specifically with dyes whose absorbance at specific wavelengths changes in response to GAG (effects due to false positives) can be eliminated, and Muco can be used accurately and efficiently. It was shown that polysaccharidosis can be selected.

[0098]

Example 3 Regarding the method 2 of the present invention (step (3)) and the method 3 of the present invention (step (4)), urine collected from 16 persons (patients or healthy subjects) was used, and the GAG composition in the urine was determined. The following experiment was conducted to confirm whether sugar analysis was possible. (1) 1 mL of pretreated urine was applied to an anion exchange column (Q-Sepharose column; manufactured by Pharmacia) to adsorb GAG in urine. The column was then loaded with 50 mM Tris-HCl buffer (pH 8.
After washing with 6), a fraction eluted with 4M NaCl was collected, desalted with PD-10 (gel filtration column; made by Pharmacia), and concentrated with a centrifugal evaporator. Water this concentrate
It was dissolved in 0.2 mL to prepare a sample solution. (2) Analysis of CS using chondroitinase ABC 40 μL of water, 70 μL of water, 100 mM Tris-HCl buffer (pH 8.
0) 40 μL and chondroitinase ABC (100 mU) were added to make a total volume of 200 μL, and after enzymatic reaction at 37 ° C. for 2 hours, the produced disaccharide was subjected to HPL using an anion exchange column.
Analyzed in C. The conditions of HPLC etc. are as follows.

Column: YMC gel PA120 S-5
(Aminopropyl group-bonded silica gel; YMC Co., Ltd.) Eluent: 0-200 mM sodium sulfate solution linear concentration gradient temperature: 25 ° C Flow rate: 0.5 mL / min Detection: Fluorescence detector (3) Chondroitinase AC-II Analysis of CS used (Analysis of DS) 70 μL of water, 100 mM sodium acetate (pH 8.
0) 40 μL, and chondroitinase AC-II (100 mU)
Was added to make the total volume 200 μL, and the enzyme reaction was carried out at 37 ° C. for 2 hours.
Analyzed by LC. The conditions of HPLC and the like are the same as those in “(2) Analysis of CS using chondroitinase ABC”.
"(2) Analysis of CS using chondroitinase ABC"
By subtracting the total amount of CS obtained here from the total amount of CS (including both CS and DS) obtained in
Was calculated. (4) Add 100 μL of the HS analysis sample solution to 100 mM sodium acetate buffer (pH 7.
0) 50 μL, 10 μM calcium acetate 50 μL, heparitinase I (2.5 mU) and heparitinase II (2.5 mU) were added to make the total volume 210 μL, and the mixture was reacted at 37 ° C. for 4 hours, and then the produced disaccharide was subjected to an anion exchange column. It was analyzed by HPLC used. The conditions of HPLC are YMC gel IE as a column.
"(2) C using chondroitinase ABC except that S (polyethyleneimine; YMC Co., Ltd.) was used.
Analysis of S ”. (5) In order to correct the amount of disaccharide obtained by the measurement analysis of creatinine with the amount of creatinine, the creatinine concentration in urine was measured using a commercially available kit (creatinine test Wako; Wako Pure Chemical Industries, Ltd.).

Table 5 shows the analysis results of urine collected from 16 persons (patients or healthy subjects).

In Table 5, "CS" indicates the amount of chondroitin sulfate, "DS" indicates the amount of dermatan sulfate,
“HS” means the amount of heparan sulfate, respectively. "MSD" means multiple sulfatase deficiency.

[0102]

[Table 5]

From Table 5, the amounts of DS and HS in the urine of the Hurler syndrome patients and the Hunter syndrome patients were high values far from those of the healthy subjects. From this result, it was confirmed that the disaccharide analysis can be used as a screening step for mucopolysaccharidosis, and it was shown that the analysis result can be associated with mucopolysaccharidosis.

[0104]

Example 4 Preparation of Kit of the Present Invention A kit of the present invention having the following constitution was prepared. 1. DMMB dye solution (15 mg / L DMMB-75 mM sodium formate buffer (pH 6.8) 1 piece 2. Heparitinase I and II (0.1U), chondroitinase ABC (U), chondroitinase ACII (U) and keratanase II Mixture containing (0.1U) 1 bottle 3. Urine dilution buffer (0.9% NaCl) 1 bottle 4. Reaction buffer (2.88M guanidine hydrochloride-0.075%)
Tween20-150mM sodium acetate buffer (pH6.8)) 1
Book 5. One microtiter plate

[0105]

INDUSTRIAL APPLICABILITY The method of the present invention is a method capable of screening a patient with mucopolysaccharidosis without omission, with accuracy, convenience, efficiency and at low cost, and is extremely highly practical.
If the possibility of mucopolysaccharidosis in all newborns can be detected by this, enzyme replacement therapy and gene therapy can be carried out at an early stage after birth when the symptoms of mucopolysaccharidosis still do not appear, and delay of mental development can be stopped. Will be more likely to be possible.

The kit of the present invention is extremely useful because it further simplifies and speeds up the method of the present invention.

The present invention can be applied not only to screening for mucopolysaccharidosis, but also to grasping the condition, determining the treatment policy, confirming the therapeutic effect, and evaluating drug development.
Extremely useful.

[Brief description of drawings]

FIG. 1 is a diagram showing a relationship (calibration curve) between a GAG concentration and OD530nm / OD590nm.

Claims (10)

[Claims] 1. A method for screening mucopolysaccharidosis, which comprises at least the following steps (1) and (2). (1) "Solution obtained by reacting urine collected from a subject with an enzyme that specifically decomposes glycosaminoglycan"
And a step of measuring the absorbance at the specific wavelength for a urine sample obtained by contacting the “urine sample with a dye whose absorbance at the specific wavelength changes by reacting with glycosaminoglycan”. (2) Calculate the amount of glycosaminoglycan decomposed by the enzyme that specifically decomposes glycosaminoglycan using the results measured in step (1), and identify the urine sample within this range Steps to take. 2. The urine collected from the subject is collected from the subject corresponding to the urine sample identified through the step including at least the following steps (A) and (B). Screening method. (A) A step of measuring the absorbance at the specific wavelength of a urine sample obtained by bringing the urine of the subject into contact with “a dye whose absorbance at the specific wavelength changes in response to glycosaminoglycan”. (B) Using the results measured in (A), the amount of the substance that reacted with the "dye whose absorbance at a specific wavelength changes in response to glycosaminoglycan" was calculated, and this was a urine sample outside the standard range. To identify. 3. At least the following step (3),
A method for screening mucopolysaccharidosis. (3) A step of analyzing a glycosaminoglycan composition in urine collected from a subject corresponding to the urine sample identified by the step (2) according to claim 1. 4. At least the following step (4) is included:
A method for screening mucopolysaccharidosis. (4) A step of associating the glycosaminoglycan composition analyzed by the step (3) of claim 3 with mucopolysaccharidosis. 5. The screening method according to claim 1, wherein the dye whose absorbance at a specific wavelength changes in response to glycosaminoglycan is dimethylmethylene blue. 6. The enzyme according to claim 1, wherein the enzyme that specifically decomposes glycosaminoglycan is one or more enzymes selected from chondroitinase, keratanase and heparitinase. Screening method. 7. The analysis of glycosaminoglycan composition is performed by causing an enzyme that specifically decomposes glycosaminoglycan to act to decompose glycosaminoglycan into a disaccharide, and analyzing the composition of the disaccharide. It is performed, The screening method of any one of Claims 3-6 characterized by the above-mentioned. 8. A screening kit for mucopolysaccharidosis, which comprises at least the following components (a) and (b): (A) Dye whose absorbance at a specific wavelength changes in response to glycosaminoglycan (b) Enzyme that specifically decomposes glycosaminoglycan 9. The screening kit according to claim 8, wherein the dye whose absorbance at a specific wavelength changes in response to glycosaminoglycan is dimethylmethylene blue. 10. The enzyme that specifically decomposes glycosaminoglycan is one or more enzymes selected from chondroitinase, keratanase and heparitinase.
The screening kit according to claim 8 or 9.