JP6989872B2 - Simultaneous screening test method for 6 diseases of inborn errors of metabolism - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Presented at the 58th Annual Meeting of the Japanese Society for Inborn Errors of Metabolism held at Keio Plaza Hotel on October 27, 2016.

Patent Law Article 30 Paragraph 2 Applicable Published in the Journal of the Japanese Society for Inborn Errors of Metabolism, Vol. 32, 58th Annual Meeting of the Japanese Society for Inborn Errors of Metabolism (issued on September 30, 2016), p. 177, P-21.

The present invention is responsible for the five diseases of lysosomal storage diseases (Fabry's disease, Pompe's disease, Gauche's disease, mucopolysaccharidosis type I (MPSI), mucopolysaccharidosis II (MPSII)) and hypophosphatase, which are inborn errors of metabolism. Regarding the test method to be screened at the same time.

Inborn errors of metabolism is a disease that develops when the activity of the enzyme required for metabolism is not expressed because the gene of the enzyme related to metabolism is congenitally deleted or mutated. It is a group of diseases that spans.

Lysosomal storage disease, which is one of inborn errors of metabolism, is a deficiency or decrease in the hydrolytic enzyme present in lysosome (also called "lysosome" or "lysosome"), which is one of the organelles. Therefore, it is also called lysosomal storage disease because it is a disease caused by the accumulation of a substrate to be decomposed in cells.

Lysosomes decompose carbohydrates and glycolipids in cells, and about 60 types of hydrolases are involved in this. When the decomposing function of lysosomes is not exerted due to the deficiency or abnormality of those responsible enzymes, substances that should be decomposed are accumulated in the body as waste products. Lysosomal storage disease is a general term for inborn errors of metabolism that occur in this way. Symptoms differ depending on the deficient enzyme, and it is known for about 30 types of diseases such as Fabry disease, Pompe disease, Gaucher disease, and mucopolysaccharidosis.

Hypophosphatase is also one of inborn errors of metabolism, and is a bone system disease caused by a gene mutation in tissue-Nonspecific Alkaline Phosphatase (hereinafter referred to as "TNSALP"). Due to TNSALP deficiency, based on the time of onset and the spread of symptoms, "perinatal type" that develops in the womb, "infant type" that develops within the second half of life, and early loss of deciduous teeth that develops in childhood It is classified into five types: "pediatric type" that accompanies it, "adult type" that develops in adulthood, and "dental limited type" whose symptoms are localized to the teeth. , Convulsions, early loss of deciduous teeth, etc. Hypophosphatosis has a high case fatality rate, and it is said that the perinatal type 1-year mortality rate is almost 100% and the infant type 1-year mortality rate is about 50%.

Lysosomal storage diseases and hypophosphatosis are different from essential amino acid metabolism disorders that can be treated with restricted intake, such as phenylketonuria, another inborn error of metabolism, and their responsible enzymes are synthesized in vivo. Therefore, gene therapy is necessary for radical cure. Currently, drugs for enzyme replacement therapy are on the market, and in many cases, early start of enzyme replacement therapy brings about clinical improvement such as improvement of patient's QOL and prevention of onset. Therefore, it is desired that these diseases are detected early by the newborn mass screening test.

In the newborn mass screening test for lysosomal storage diseases, a method of measuring enzyme activity by a synthetic substrate method is used using a filter paper soaked with blood and dried (hereinafter referred to as "dry blood filter paper"). In addition, enzyme activity measurement methods using tandem analyzers and pilot studies using microchannel measurement kits that require specialized equipment but can be measured quickly are being carried out, which is useful for newborn mass screening tests. Gender is shown.

On the other hand, for hypophosphatosis, the screening test using dry blood filter paper has not been performed because the enzyme activity of TNSALP cannot be specifically measured by the existing measurement method.

Alkaline phosphatase (hereinafter referred to as "ALP") in the living body is abundant in the order of small intestinal mucosa, placenta, mammary gland, kidney, bone, lung, liver, and spleen, and is abundant in skeletal muscle and connective tissue. ALP enzyme activity in serum is derived from ALP produced in these tissues. ALP is roughly classified into bone type and liver type, which are non-organ specific types, and small intestine type, placental type, and embryonic cell type ALP, which are kidney type and organ specific. There are 6 types of ALP isozymes in serum ALP, and they are measured as the total ALP enzyme activity.

In particular, in recent years, bone-type alkaline phosphatase (BAP; Bone Specific Alkaline Phosphatase) has attracted attention as a bone formation marker, and an enzyme-linked immunosorbent assay (ELISA method) using a BAP-specific antibody for specifically measuring BAP in serum has been used. A chemical luminescent enzyme immunoassay (CLEIA method) has been developed and is commercially available as a measurement kit. However, these measurement kits are expensive and require a dedicated automated analyzer, which makes them unsuitable for mass screening tests.

Hypophosphatosis is caused by a decrease in the enzymatic activity of TNSALP due to an abnormality in the TNSALP gene. Therefore, it can be easily determined by confirming by measurement that the total ALP enzyme activity in serum is decreased.

The enzymatic activity of ALP in serum is generally measured by a measurement method using para-nitrophenyl phosphate (p-NPP) as a substrate and using a biochemical analyzer. However, it is easily affected by hemoglobin and other blood pigments, and is not suitable for measurement with whole blood samples or hemolyzed samples. Further, as a more sensitive substrate, there is also an enzyme activity method using a fluorescent substrate. Although the method using 4-Methylumbelliferyl phosphate (hereinafter referred to as "4MU-Phos"), which is a synthetic substrate, as a substrate is widely used as a substrate for ALP enzyme activity produced by microorganisms and enzyme immunoassay. Since 4MU-P causes self-decomposition by divalent metal ions such as iron (II) ion (Fe ²⁺ ), it is not possible to measure a sample in which hemoglobin coexists.

Magnesium ion (Mg ²⁺ ) is essential for the expression of enzyme activity of ALP, and coexistence of Mg ²⁺ is essential for ALP measurement. Therefore, when measuring a sample containing a large amount of Fe ²⁺ such as hemoglobin, It is necessary to coexist with an additive that does not interfere with the action of Mg ²⁺ and masks metal ions such as Fe ²⁺ that act on the self-decomposition of the substrate.

Patent Document 1 describes methods and kits for assaying the activity of lysosomal enzymes present in dried body fluids and cell tissue samples. The methods and kits described in Patent Document 1 are assayed for various lysosomal storage disorders individually for each disease.

Patent Document 2 describes multiple screening of lysosomal storage disease (LDS) including MPSI and MPSII. Patent Document 2 relates to a compound, a reagent, and a method for identifying and quantifying a plurality of target enzymes and proteins. It is based on the included multiplexed bead technology.

Patent Document 3 describes a method for testing α-L-Iduronidase (IDU) enzyme activity and a method for screening newborns for MPSI. The method described in Patent Document 3 comprises extracting an aqueous enzymatic reaction mixture containing IDU, IDU product and IDU internal standard with an organic solvent in order to provide an organic phase containing IDU product and IDU internal standard, and IDU. Includes steps to determine the amount of product. In Patent Document 3, only the IDU enzyme is the subject of the assay alone.

In Patent Document 4, heparan sulfate in a blood sample derived from an animal having mucopolysaccharidosis type I, type II, type III or type VII may be measured, and the measurement result and mucopolysaccharidosis I A method for detecting mucopolysaccharidosis type I, type II, type III or type VII is described, which comprises at least a step of associating with type, type II, type III or type VII. The method described in Patent Document 4 is a method for detecting lysosomal disease, which comprises at least a step of measuring a single type of glycosaminoglycan (GAG) in a sample and associating the measurement result with lysosomal disease.

Iduronate-2-sulfatase (IDS) As a highly sensitive method for measuring enzyme activity, an enzyme activity method using a fluorescent substrate is generally used, and the synthetic substrate 4-Methylumbelliferyl-α-L-Idopyranosiduronic Acid- A method is known in which 2-Sulfate and Sodium Salt (hereinafter abbreviated as "4MU-IDS") are used as a substrate and measured by a two-step enzymatic reaction to which Iduronidase is added. However, since this synthase is very expensive and requires a measurement time of 48 hours or more, cheaper, simpler and faster measurement is required for use in a newborn mass screening test.

In Patent Document 5, as a test method for hypophosphatosis, the diagnosis of hypophosphatosis is made by examining the presence or absence of gene mutation by melting curve analysis of the gene amplification cross section of a specific site using 12 pairs of primers for the TNSALP gene. How to do this is described.

Patent Document 6 is a rapid mass screening test method for a lysosome disease-responsible enzyme that rapidly measures the enzyme activity of IDU and IDS from a single dry blood filter, and (1) blood from a dry blood filter using a common extract. It includes a step of extracting a sample, (2) a step of adding a substrate solution containing a synthetic substrate to the extracted blood sample to carry out an enzymatic reaction, and (3) a step of measuring fluorescence of the product obtained by the enzymatic reaction. The method is described.

Patent Document 7 describes a rapid measurement of lysosomal storage disease-responsible enzyme for rapidly measuring α-D-galactosidase A (GLA), acidic α-glucosidase (GAA), and acidic β-glucosidase (GBA) from a single dry blood filter. In the mass screening test method, (1) a step of extracting a blood sample from a dry blood filter using a common extract, and (2) a step of adding a substrate solution containing a synthetic substrate to the extracted blood sample to cause an enzymatic reaction. , (3) A method including a step of fluorescently measuring a product obtained by an enzymatic reaction is described.

Non-Patent Document 1 describes that the measurement of the activity of GAL, which is the enzyme responsible for Fabry disease, using dry blood filter paper was effective for the screening of Fabry disease in newborns. In Non-Patent Document 1, only the GAL enzyme is the object of measurement alone.

In Non-Patent Document 2, measurement of the activity of GAA, which is a Pompe disease responsible enzyme in dry blood filter paper and fibroblasts, is effective for Pompe disease screening in newborns, and a method for avoiding the influence of hemoglobin in a sample. It is described that the precipitation removal method using barium hydroxide and zinc sulfate was useful. In Non-Patent Document 2, only the GAA enzyme is the subject of measurement alone.

In Non-Patent Document 3, multiple screening of lysosomal storage disease (LDS) including Fabry disease, Pompe disease, and Gaucher disease using a digital microchannel measuring device and a dedicated kit is used in a pilot study of newborn mass screening. It is stated that there is.

Non-Patent Document 4 describes that the assay of IDU, which is the responsible enzyme of MPSI, was effective for MPSI screening in newborns. In Non-Patent Document 4, only the IDU enzyme is the subject of the assay alone.

Non-Patent Document 5 describes that the enzyme activity of IDS, which is the responsible enzyme of MPSII, was measured in leukocytes. In Non-Patent Document 5, only IDS is the subject of the assay alone.

Non-Patent Document 6 describes that the TNSALP enzyme, which is the responsible enzyme for hypophosphatosis, was measured by the ELISA method. Non-Patent Document 5 shows that the enzyme activity and the amount of antigen in the serum of a patient with hypophosphatase are lower than those of a normal person.

Non-Patent Document 7 describes that the serum alkaline phosphatase (total alkaline phosphatase) activity in patients with hypophosphatase was 1/5 or less of the normal value (serum) in children.

U.S. Pat. No. 7,563,591 Special table 2006-523300 Special table 2010-536347 JP 2013-11621 JP 2011-50283 JP-A-2017-169528 JP-A-2017-169529

Journal of Human Genetics (2013) 58, 548-552 Molecular Genetics and Metabolism 103 (2011) 12-17 Dried Blood Spots: Application and Techniques, 2014,325-331 Orphanet Journal of Rare Disease, 8, 147, 2013 Clinical Biochemistry, 47, 1297-1299, 2014 Jpn.J Clin Chem 23, 203-208, 1994 Oral Disease Magazine 58 (3), 613-623, 1991

The present invention measures the responsible enzyme activity of five lysosomal storage diseases (Fabry's disease, Pompe's disease, Gaucher's disease, mucopolysaccharidosis type I, mucopolysaccharidosis II), which are inborn errors of metabolism in neonatal mass screening tests. It is an object of the present invention to provide a simple, quick and inexpensive method for carrying out the disease without the need for large-scale measuring equipment or dedicated equipment.

In the newborn mass screening test, a sample extracted from dry blood filter paper is used as a measurement sample. In the conventional measurement method, the composition of the extract differs depending on the responsible enzyme to be inspected, and only one type of enzyme activity can be measured from one dry blood filter paper. To measure multiple enzymes, multiple sheets are used. A dry blood filter paper was needed. In addition, in order to measure the enzyme activity of a sample extracted from dried blood filter paper, the reaction time differs depending on the enzyme to be measured, and it is a complicated task to measure the enzyme activity of multiple enzymes at once from the same sample. I needed it.

Furthermore, in the conventional measurement method, the measurement wavelength, operating procedure, reaction time, extract composition, and reaction stop solution composition differ depending on the responsible enzyme to be measured. Therefore, in order to measure a large number of samples at the same time, the responsible enzyme An analyst was needed for each.

As a result of diligent studies to solve the above problems, the present inventors have studied Fabry disease-responsible enzyme, Pompe disease-responsible enzyme, Gauche disease-responsible enzyme, mucopolysaccharidosis type I-responsible enzyme, mucopolysaccharidosis type II-responsible enzyme, We have found the composition of an extract that can be used in common (hereinafter referred to as "common extract") without affecting the decrease in enzyme activity during extraction of the enzyme responsible for hypophosphatosis and the pH at the time of measuring enzyme activity. Furthermore, they have found the composition of a reaction terminator (hereinafter referred to as "common reaction terminator") that can measure the extracted sample with high sensitivity and can be used in common with each substrate solution composition.

We also found that by shifting the measurement wavelength of enzyme activity, the influence of fluorescent substances and fluorescence inhibitors in the sample extracted from dried blood filter paper was reduced, and the sample was centrifugally operated, which was necessary before the measurement. Is unnecessary.

Furthermore, we have found that the enzyme activity of each enzyme per unit time decreases as the enzyme reaction time increases. Furthermore, by setting the enzyme reaction time to a reaction time that can be commonly used for the six enzyme activities, the complexity of the measurement operation is eliminated, and the lysosome enzyme activity measurement can be sufficiently measured even in a shorter time than before. By finding common conditions (sample liquid amount, substrate liquid amount, reaction stop liquid amount, enzyme reaction time), even one analyst can measure multiple items at the same time.

Based on these findings, from a sample extracted from a single dry blood filter, Fabry's disease-responsible enzyme, Pompe's disease-responsible enzyme, Gauche's disease-responsible enzyme, mucopolysaccharidosis type I-responsible enzyme, mucopolysaccharidosis-type II-responsible enzyme, And, the present invention has been completed by finding a method for simultaneously, highly sensitive, simple, rapid, efficient and inexpensive detection of enzyme activity measurement of the enzyme responsible for low phosphatosis.

That is, the present invention is a method for simultaneously detecting 6 types of inborn errors of metabolism by measuring the enzyme activity of a sample extracted from one dry blood filter paper using 6 types of substrate solutions, and the test method thereof. Provides a kit for use in. Here, the inborn errors of metabolism in the present invention are six diseases of Fabry's disease, Pompe's disease, Gauche's disease, mucopolysaccharidosis type I, mucopolysaccharidosis type II, and hypophosphatase.

Therefore, the present invention includes:
[1] A rapid mass screening test method for inborn errors of metabolism, which rapidly measures the activity of enzymes responsible for multiple inborn errors of metabolism from a single sheet of dry blood filter paper.
(Group A) α-D-galactosidase A (GLA), acidic α-glucosidase (GAA), acidic β-glucosidase (GBA)
(Group B) α-L-Iduronidase (IDU), Iduronate-2-sulfatase (IDS) and (Group C) Tissue non-specific alkaline phosphatase (TNSALP)
Enzymes of 1 or more in each group selected from at least two groups of, comprising the following steps:
(1) A step of extracting a blood sample from a dry blood filter paper using a common extract.
(2) A step of adding a substrate solution containing a synthetic substrate to an extracted blood sample to cause an enzymatic reaction.
(3) A step of measuring fluorescence of a product obtained by an enzymatic reaction.
[2] The method according to [1], wherein the common extract is a buffer solution containing no phosphate ion and citrate ion.
[3] The method according to [1] or [2], wherein the common extract contains a surfactant.
[4] The method according to [3], wherein the surfactant is Triton X-100 and is contained in an amount of 0.05% to 0.5%.
[5] The item according to any one of [1] to [4], wherein the common extract contains 0.02% to 0.1% sodium azide and 0.05% to 0.5% bovine serum albumin (BSA). Method.
[6] When the substrate solution is the responsible enzyme of Group A, (i) a buffer solution containing N-acetylgalactosamine and a 4-Methylumbelliferyl synthetic substrate, (ii) a buffer solution containing acarbose and a 4-Methylumbelliferyl synthetic substrate, And (iii) a combination of buffers containing 4-Methylumbelliferyl synthetic substrate, and in the case of Group B responsible enzymes, (iv) buffers containing potassium chloride and 4-Methylumbelliferyl synthetic substrate, and (v) Iduronidase and 4 -A combination of buffers containing a Methylumbelliferyl synthetic substrate, and in the case of the responsible enzyme of Group C, (vi) a buffer containing L-phenylalanine and a 4-Methylumbelliferyl synthetic substrate, any of [1] to [5]. The method described in item 1.
[7] The method according to [6], wherein the 4-Methyum belliferyl synthetic substrate in (i) above is 4-methylum-belliferyl-α-D-galactopyranoside and is contained at 1 mM to 10 mM.
[8] The method according to [6] or [7], wherein the pH of the substrate solution in (i) above is 4.0 to 5.0.
[9] The substrate solution in (i) above is a citric acid-sodium phosphate buffer solution containing 0.1 mM to 1.0 mM Dithiothreitol (DTT), according to any one of [6] to [8]. the method of.
[10] The 4-Methyum belliferyl synthetic substrate in (ii) above is 4-methylum-belliferyl-α-D-glucopyranoside, which is contained at 1 mM to 10 mM and acarbose at 1 μM to 10 μM, according to [6]. the method of.
[11] The method according to [6] or [10], wherein the pH of the substrate solution in (ii) is 3.5 to 4.5.
[12] The substrate solution in (ii) above is a citric acid-potassium phosphate buffer solution containing 0.1 mM to 1.0 mM Dithiothreitol (DTT), according to [6], [10] or [11]. Method.
[13] The method according to [6], wherein the 4-Methyum belliferyl synthetic substrate in (iii) is 4-methylum-belliferyl-β-D-glucopyranoside, which is contained in an amount of 1 mM to 10 mM.
[14] The method according to [6] or [13], wherein the pH of the substrate solution in (iii) is 4.5 to 5.5.
[15] The method according to [6], [13] or [14], wherein the substrate solution in (iii) is a citric acid-sodium phosphate buffer solution.
[16] The method according to any one of [6] and [13] to [15], wherein the substrate solution in (iii) above contains sodium taurodeoxycholate as a similar enzyme inhibitor.
[17] The method according to any one of [6] and [13] to [16], wherein the substrate solution in (iii) contains 0.1 mM to 1 mM Dithiothreitol (DTT).
[18] The method according to [6], wherein the 4-Methyumbelliferyl synthetic substrate in (iv) is 4-Methylumbelliferyl α-L-Idopyranosiduronic Acid, Sodium Salt, which is contained at 0.1 mM to 3 mM.
[19] The method according to [6] or [18], wherein the pH of the substrate solution in (iv) is 3 to 4.
[20] The method according to any one of [18] to [19], wherein the buffer solution of the substrate solution in (iv) has a reducing action.
[21] The method according to [20], wherein the buffer solution is a sodium formate buffer solution.
[22] The method according to any one of [6] to [22], wherein the substrate solution in (iv) contains 0.1 mM to 1 mM Dithiothreitol (DTT).
[23] The method according to any one of [6] to [11], wherein the substrate solution in (iv) contains 0.01% to 0.1% of Polyoxyethyleneglycol Dodecyl Ether (Brij-35).
[24] Any one of [6] to [24], wherein the substrate solution in (iv) contains 1 μg / mL to 20 μg / mL of D-Saccharic acid 1,4-lactone as a similar enzyme inhibitor. The method described in.
[25] The method according to [6], wherein the Iduronidase in (v) above is an Iduronidase produced by a genetic recombination technique and is contained at 0.1 μg / mL to 2 μg / mL.
[26] The 4-methylumbelliferyl synthetic substrate in (v) above is 4-Methylumbelliferyl-aL-Idopyranosiduronic Acid-2-Sulfate, Sodium Salt, which is contained at 0.1 mM to 3 mM, according to [6] or [25]. the method of.
[27] The method according to [6], [25] or [26], wherein the pH of the substrate solution in (v) is 4 to 5.
[28] The method according to any one of [6] or [25] to [27], wherein the buffer solution of the substrate solution in (v) is a sodium acetate buffer solution.
[29] The method according to any one of [6] or [25] to [28], wherein the substrate solution in (v) contains 10 mM to 30 mM lead acetate or 2 to 20 mM cerium acetate.
[30] The method according to [6] or [29], wherein the 4-methylumbelliferyl synthetic substrate in (vi) is 4-Methylumbelliferylphosphate and is contained at 0.05 mM to 2 mM.
[31] The method according to [6], [29] or [30], wherein the pH of the substrate solution in (vi) is 9 to 11.
[32] The method according to any one of [6] or [29] to [31], wherein the buffer solution of the substrate solution in (vi) is a Tris buffer solution.
[33] The method according to any one of [6] or [30] to [32], wherein the substrate solution in (vi) contains 1 mM to 30 mM L-phenylalanine.
[34] The method according to any one of [6] or [30] to [33], wherein the substrate solution in (vi) contains 0.5 mM to 2 mM MgCl ₂ as an enzyme activity enhancer.
[35] The method according to any one of [6] or [30] to [34], wherein the substrate solution in (vi) contains 1 mM to 30 mM sodium succinate as a substrate autolysis inhibitor.
[36] The item according to any one of [1] to [35], wherein the enzyme reaction is stopped by adding a common reaction stop solution containing glycine and EDTA to the enzyme reaction solution in the step (2). Method.
[37] The method according to [36], wherein the pH of the common reaction terminator is 10 to 11.
[38] The method according to [36] or [37], wherein the enzymatic reaction is carried out at a temperature of 25 to 45 ° C., and then the common reaction terminator is added.
[39] The method according to any one of [1] to [39], wherein the enzymatic reaction is carried out in 1 to 4 hours.
[40] The method according to any one of [1] to [39], wherein the measurement wavelength of the fluorescence measurement is an excitation wavelength of 365 to 375 nm and a detection wavelength of 460 to 470 nm.
[41] A method for simultaneously extracting a plurality of responsible enzymes for inborn errors of metabolism from one sheet of dry blood filter paper, which comprises using a common extract.
(Group A) α-D-galactosidase A (GLA), acidic α-glucosidase (GAA), acidic β-glucosidase (GBA)
(Group B) α-L-Iduronidase (IDU), Iduronate-2-sulfatase (IDS) and (Group C) Tissue non-specific alkaline phosphatase (TNSALP)
A method, which is one or more enzymes in each group, selected from at least two groups of.
[42] The method according to [41], wherein the common extract is a buffer solution containing no phosphate ion and citrate ion.
[43] The method according to [41] or [42], wherein the common extract contains a surfactant.
[44] The method according to [43], wherein the surfactant is TronX-100 and is contained in an amount of 0.05% to 0.5%.
[45] The item according to any one of [41] to [44], wherein the common extract contains 0.02% to 0.1% sodium azide and 0.05% to 0.5% bovine serum albumin (BSA). Method.
[46] A kit for use in the inspection method according to [1], including:
(1) Dry blood filter paper,
(2) Common extract,
(3) Substrate solution containing synthetic substrate,
(4) A common reaction terminator and (5) a means for measuring fluorescence of a product obtained by an enzymatic reaction.
[47] The kit according to [46], wherein the common extract is purified water containing 0.1% BSA, 0.1% Trtion X-100 and 0.05% sodium azide.
[48] The substrate solution containing the synthetic substrate is a substrate solution for measuring GLA enzyme activity for detecting Fabry's disease, a substrate solution for measuring GAA enzyme activity for detecting Pompe's disease, and a substrate solution for detecting Gaucher's disease. GBA enzyme activity measurement substrate solution, IDU enzyme activity measurement MPSI substrate solution for detecting mucopolysaccharidosis type I (MPSI), IDS enzyme activity measurement for mucopolysaccharidosis II (MPSII) detection The substrate solution for measuring GLA enzyme activity for detecting Fabry's disease, which is a combination of MPSII substrate solution and TNSALP substrate solution for detecting hypophosphatase disease, is 3.0 mM 4-methylumbelliferyl-α-D-garactopyranoside. And 100 mM citrate-200 mM sodium phosphate buffer at pH 4.4 containing 100 mM N-acetyl-D-galactosamine, 0.5 mM DTT; 2.0 mM 4 substrate solution for GAA enzyme activity measurement to detect Pompe disease -250 mM citrate-250 mM potassium phosphate buffer at pH 4.0 containing methylumbelliferyl-α-D-glucopyranoside and 4.5 μM acarbose, 0.5 mM DTT; substrate solution for GBA enzyme activity measurement to detect Gauche disease 100 mM citrate-100 mM sodium phosphate buffer with pH 5.0 containing 3.0 mM 4-methylumbelliferyl-β-D-glucopyranoside, 0.5 mM DTT and 0.3% sodium taurodeoxycholate; MPS I substrate solution is 0.6 mM 4- Methylumbelliferyl-α-L-Idopyranosiduronic acid, sodium salt, 5 μg / mL D-Saccharic acid 1,4-lactone, 0.05% Birij-35, 50 mM KCl and 0.25 mM DTT containing 100 mM sodium formate buffer at pH 3-4; MPSII PH 4 substrate solution containing 0.13 mM 4-methylumbelliferyl-α-L-Idopyranosiduronic Acid -2-sulfate, Sodium Salt (4MU-IDS), 1 μg / mL recombinant Iduronidase and 20 mM lead acetate or 2-20 mM cerium acetate .5 50 mM sodium acetate buffer; TNSALP substrate solution is 0 The kit of claim 46 or 47, which is a pH 9-11 100 mM Tris buffer containing .2 mM 4-methylumbelliferyl-phosphate, 0.05% TritonX-100, 10 mM sodium succinate, 10 mM L-phenylalanine, 1 mM MgCl ₂ .
[49] The kit according to any one of [46] to [48], wherein the common reaction terminator is a 300 mM glycine-NaOH buffer (pH 10.6) containing 10 mM EDTA.

The present invention presents 6 diseases of inborn errors of metabolism (Fabry's disease, Pompe's disease, Gauche's disease, mucopolysaccharidosis type I) from one dry blood filter in a newborn mass screening test in which the number of dry blood filters is limited. , Mucopolysaccharidosis type II, hypophosphatase).

In the present invention, by standardizing the measurement conditions, a large number of samples can be measured at one time, the time required for the test can be significantly shortened, and a plurality of items can be tested at the same time.

INDUSTRIAL APPLICABILITY According to the present invention, it has become possible to use the dry blood filter paper used for existing neonatal metabolic disorders as it is for newborn babies whose blood as a sample is difficult to collect. If the possibility of 6 new inborn errors of metabolism can be detected in all newborns, enzyme replacement therapy, gene therapy, bone marrow transplantation, etc. can be performed at an early stage after birth when clinical symptoms have not yet appeared. , There is a high possibility that delays in mental development can be stopped.

Further, the present invention is applied not only to the detection of inborn errors of metabolism in a mass screening test for newborns, but also to grasping the pathological condition, determining the treatment policy, confirming the therapeutic effect, follow-up, monitoring, evaluation of drug development, and the like. can do.

The relationship between the excitation wavelength (300 to 400 nm) and the detection sensitivity (S / N ratio) is shown. The relationship between the detection wavelength (400 to 500 nm) and the detection sensitivity (S / N ratio) is shown. The S / N ratio when GLA is measured with a combination of multiple measurement wavelengths is shown. The S / N ratio when GAA is measured with a combination of multiple measurement wavelengths is shown. The S / N ratio when GBA is measured with a combination of multiple measurement wavelengths is shown. The results of comparing the enzyme activity values of the patient sample and the normal human sample with and without acarbose are shown. The comparison result of GLA, GAA, GBA enzyme activity with and without DTT is shown. The results of fluorescence measurement (with or without DTT) without the 4MU synthetic substrate in the substrate solution are shown. The S / N ratio in the inspection method according to the present invention and the inspection method (individual measurement conditions) according to the prior art document is shown. The comparison result of the additive which affects the IDU enzyme activity is shown. The comparison result of the substrate solution KCl concentration on the IDU enzyme activity is shown. The comparison result of the buffer solution (formic acid buffer solution and acetate buffer solution) of the substrate solution affecting the IDU enzyme activity is shown. The effect of the combination of IDS enzyme activity and extraction buffer is shown. The relationship between IDS enzyme activity and substrate solution pH is shown. The IDS enzyme activity and the effect of adding cerium acetate and lead acetate are shown. The comparison result of the metal chelating agent of TNSALP enzyme activity is shown. The comparison result of TNSALP enzyme activity and the concentration of Na citrate and Na succinate is shown. The comparison result of TNSALP enzyme activity and L-phenylalanine concentration is shown. The relationship between GLA, GAA, GBA, MPSI, MPSII enzyme activity and enzyme reaction time is shown. The relationship between the TNSALP enzyme activity and the enzyme reaction time is shown. It is a flowchart exemplifying the enzyme activity measurement method in this invention. The distribution map of GLA enzyme activity in the dry blood filter paper of a newborn baby is shown. Arrows indicate GLA enzyme activity in dry blood filter paper of Fabry patients. The distribution map of GAA enzyme activity in the dry blood filter paper of a newborn baby is shown. Arrows indicate GAA enzyme activity in dry blood filter paper of Pompe disease patients. The GBA enzyme activity distribution map in the dry blood filter paper of a newborn is shown. Arrows indicate GBA enzyme activity in dry blood filter paper of patients with Gaucher's disease. The IDU enzyme activity distribution map in the dry blood filter paper of a newborn is shown. Arrows indicate IDU enzyme activity in dry blood filter paper of patients with MPSI disease. The IDS enzyme activity distribution map in the dry blood filter paper of a newborn is shown. Arrows indicate IDS enzyme activity in dry blood filter paper of MPS II patients. The distribution map of TNSALP enzyme activity in the dry blood filter paper of a newborn baby is shown. Arrows indicate IDS enzyme activity in dry blood filter paper for patients with hypophosphatase.

(Detailed description of the invention)
Enzyme activity measurement for congenital metabolic disorders using dried blood filter paper as a sample can be performed by directly mixing the dried blood filter paper and the substrate solution to cause an enzymatic reaction, or by adding a small amount of buffer to the dried blood filter paper and then using the substrate. There are a method of adding a liquid to cause a reaction, a method of extracting blood from a dry blood filter paper, and a method of adding a substrate liquid to the extracted blood sample to cause an enzymatic reaction.

The present invention comprises, from a single dry blood filter, Fabry disease responsible enzyme (GLA), Pompe disease responsible enzyme (GAA), Gauche disease responsible enzyme (GBA), mucopolysaccharidosis type I (MPSI) responsible enzyme (IDU), Blood containing mucopolysaccharidosis type II (MPSII) responsible enzyme (IDS) and hypophosphatase disease responsible enzyme (TNSALP) was extracted, and the extracted blood sample was used to measure the activity of 6 types of enzymes in dry blood filter paper. How to do it.

The measurement by the method of the present invention consists of (1) a step of extracting a blood sample from a dry blood filter paper using a common extract, and (2) adding a substrate solution containing a synthetic substrate to the extracted blood sample to cause an enzymatic reaction. The process is completed by three steps: (3) a step of measuring the fluorescence of the product obtained by the enzymatic reaction.

In the enzyme activity measurement of a blood sample, the buffer capacity of the optimum pH is set in either one or both of the extract and the substrate solution so that the pH is suitable for the enzyme activity measurement when the extracted blood sample and the substrate solution are mixed. It was necessary to have a composition to have, and in the known measurement method, the extract was different for each enzyme to be measured. Therefore, a separate dry blood filter paper is required for each enzyme to be measured, and a plurality of dry blood filter papers are required to measure a plurality of items.

In the present invention, we have found a common extract composition that can effectively extract each enzyme from dry blood filter paper at the same time without interfering with the measurement of enzyme activity of 6 kinds of enzymes, and multiple enzymes from one dry blood filter paper. Was made possible to measure at the same time.

The common extract according to the method of the present invention is preferably a low buffering solution that does not inhibit the optimum pH of the enzyme reaction when mixed with each substrate solution, and more preferably phosphoric acid that interferes with the reaction of sulfatase enzyme or phosphatase enzyme. It is a buffer solution that does not contain ions or citrate ions.

The common extract according to the method of the present invention does not contain ions that interfere with the enzymatic reaction of GLA, GAA, GBA, IDU, IDS and TNSALP, and in order to stabilize the extracted enzyme, bovine serum albumin (hereinafter, "BSA") It is preferable to allow a dilute protein such as () to coexist and have a pH buffering ability. In addition, since the lysosome enzyme to be measured is present in lysosome in blood cells such as leukocytes, a surfactant (TritonX-100, etc.) having the effect of crushing the cells and releasing the enzyme in the extract is allowed to coexist. Is more preferable. On the other hand, since TNSALP is present on the cell surface in blood or in serum, it is preferable to coexist with a surfactant in order to uniformly release the enzyme in the extract.

The common extract according to the present invention does not work to suppress the activity of the enzyme to be measured, and in the process of extracting the enzyme, an additive having an effect of suppressing a non-specific reaction due to an enzyme activity other than the intended purpose coexists, and dry blood is used. It is preferable to coexist with a surfactant that promotes extraction from the filter paper. Since the surfactant may suppress or enhance the enzyme reaction at a concentration that generally acts, it is necessary to add the surfactant at a concentration that does not inhibit the enzyme activity.

The common extract of the present invention contains 0.05% to 0.5% BSA, 0.05% to 0.5% Trtion X-100 and 0.02% to 0.1% sodium azide. The composition of the common extract of one embodiment of the present invention is "purified water containing 0.1% BSA, 0.1% Trtion X-100 and 0.05% sodium azide". BSA in the common extract has a weak pH buffering capacity in the common extract, and also acts on the enhancement of IDU enzyme activity and the stabilization of each enzyme. Triton X-100, a surfactant, promotes the elution of blood components from dry blood filter paper and the elution of enzymes by blood cell disruption, and is added to shorten the extraction time. Sodium azide is added to prevent non-specific reactions caused by peroxidases present in blood and to suppress preservatives in the common extract. The concentration of each additive is preferably set as a concentration that does not suppress each other's enzyme activity.

As the substrate solution according to the present invention, by using a buffer solution having a buffering capacity in a pH range suitable for measuring each enzyme activity, it is possible to measure each enzyme using a blood sample extracted with a common extract.

The synthetic substrate used in the substrate solution needs to be a substrate having a structure that serves as a specific substrate for the enzyme to be measured. By using such a synthetic substrate, the reaction specificity with the enzyme to be measured can be improved. However, since there are innumerable enzymes that carry out similar reactions in the extracted blood sample, it is preferable to coexist with a similar enzyme inhibitor for the purpose of further increasing the specificity.

The substrate solution (hereinafter referred to as "GLA substrate solution") used for measuring the GLA enzyme activity for detecting Fabry disease is 1 mM to 10 mM 4-methylumbelliferyl-α-D-garactopyranoside (hereinafter referred to as "4MU-α-"). It is abbreviated as "GAL") and contains 10 mM to 200 mM N-acetyl-D-galactosamine (hereinafter abbreviated as "GalNAc"). The composition in one embodiment of the GLA substrate solution is "100 mM citric acid-200 mM Na phosphate buffer with pH 4.4 containing 3.0 mM 4MU-α-GAL and 100 mM GaLNAc, 0.5 mM DTT". The pH may be 4.0 to 5.0.

The substrate solution (hereinafter referred to as "GAA substrate solution") used for measuring GAA enzyme activity for detecting Pompe disease is 1 mM to 10 mM 4-methylumbelliferyl-α-D-glucopyranoside (hereinafter referred to as "4MU-α-"). It is abbreviated as "Glu") and contains 1 μM to 10 μM acarbose. The composition in one embodiment of the GAA substrate solution is "250 mM citric acid-250 mM potassium phosphate buffer at pH 4.0 containing 2.0 mM 4MU-α-Glu, 4.5 μM acarbose, 0.5 mM DTT". The pH may be 3.5 to 4.5. Acarbose is added to inhibit the action of α-glucosidases other than GAA contained in blood samples.

In the measurement of GAA, it is necessary to improve the specificity of GAA enzyme activity measurement by inhibiting α-glucosidase other than GAA in blood by adding an α-glucosidase inhibitor (acarbose). However, the use of high concentrations of acarbose also affects the activity of the GAA enzyme. Therefore, in the present invention, the dry blood filter paper of a Pompe patient and the dry blood filter paper of a normal person are used for comparative verification with a substrate solution containing or without acarbose, and the concentration of acarbose is determined.

The substrate solution (hereinafter referred to as "GBA substrate solution") used for measuring GBA enzyme activity for detecting Gaucher's disease is 1 mM to 10 mM 4-methylumbelliferyl-β-D-glucopyranoside (hereinafter referred to as "4MU-β-"). Glu ”) and 0.1% to 1% sodium taurodeoxycholate (hereinafter abbreviated as“ TAUDA ”). The composition in one embodiment of the GBA substrate solution is "100 mM citric acid-100 mM Na phosphate buffer at pH 5.0 containing 3.0 mM 4MU-β-Glu, 0.3% TAUDA, 0.5 mM DTT". The pH may be 4.5 to 5.5.

Sodium taurocholate (hereinafter abbreviated as "TAUA") is generally used as a similar enzyme inhibitor added to the substrate solution for Gaucher's disease, but TAUDA is more effective than TAUA in enhancing GBA enzyme activity. By using, the GBA enzyme activity of a low-concentration sample can be measured. Since GBA enzyme is abundant on the membrane of lysosome, changing to TAUDA similar to deoxycholic acid, which is highly effective in solubilizing membrane proteins, is effective in enhancing GBA enzyme activity measurement. ..

The substrate solution used for measuring IDU enzyme activity to detect MPSI (hereinafter referred to as "MPSI substrate solution") is 0.1 mM to 3 mM 4-methylumbelliferyl-α-L-Idopyranosiduronic acid, sodium salt (hereinafter referred to as "sodium salt"). 4MU-IDUA ”), 1 μg / mL to 20 μg / mL D-Saccharic acid 1,4-lactone (hereinafter abbreviated as“ D-Sac ”), 0.01% to 0.1% Birij-35 (Polyoxyethyleneglycol) Dodecyl Ether), containing 10 mM to 100 mM KCl (potassium chloride) and 0.1 mM to 1.0 mM DTT (Dithiothreitol). The composition of the MPSI substrate solution in one embodiment is "100 mM Na formic acid buffer of pH 3-4 containing 0.6 mM 4MU-IDUA, 5 μg / mL D-Sac, 0.05% Birij-35, 50 mM KCl and 0.25 mM DTT". be.

In the MPSI substrate solution having the above composition, KCl, Brij-35 as a surfactant, and an additive of DTT are added at the optimum concentration for the effect of enhancing the IDU enzyme activity. In addition, 4MU-IDUA, which is a synthetic substrate, has improved reactivity with IDU by using sodium salt, which enables highly sensitive measurement.

An example of a similar enzyme inhibitor added to the MPSI substrate solution is D-Sac. Similar enzyme inhibitors inhibit various enzymes having similar actions other than IDU, and act to enhance the specificity of IDU enzyme activity measurement. The concentration may be 1 μg / mL or more, and 5 μg / mL to 10 μg / mL can completely inhibit the non-specific enzymatic reaction by a similar enzyme.

Since the Base buffer solution of the MPSI substrate solution causes an enzymatic reaction on the acidic side of pH 3 to 4, a formic acid buffer solution having a reducing action is preferable, and a formic acid Na buffer solution having a pH of 3.5 is more preferable.

On the other hand, the substrate solution used for measuring the IDS enzyme activity for detecting MPSII (hereinafter referred to as "MPSII substrate solution") is a 4MU-IDS of 0.1 mM to 3 mM, and a gene set of 0.1 μg / mL to 2 μg / mL. It contains a substitute Iduronidase (hereinafter abbreviated as "r-IDU") and 10 mM to 30 mM lead acetate or 2 to 20 mM cerium acetate. The composition in one embodiment of the MPSII substrate solution is "0.125 mM 4MU-IDS, 0.7 μg / mL r-IDU and 5 mM cerium acetate in pH 4.5 50 mM Na acetate buffer". The pH of the buffer may be 4-5.

The MPSII substrate solution in the above composition was devised as a composition that enables an enzymatic reaction that previously required two steps in one step. In the IDS enzymatic reaction using a known 4MU-IDS substrate, IDS and 4MU-IDS are reacted as the first enzymatic reaction to produce 4MU-IDUA, which is a substrate for Iduronidase. Then, after stopping the IDS enzyme reaction by adding phosphate buffered saline or the like, Iduronidase is added to decompose 4MU-IDUA, which is a fluorescent substance 4-methylumbelliferone (hereinafter abbreviated as "4MU"). It is necessary to carry out a second-stage enzymatic reaction to release. The MPSII substrate solution in the present invention has a composition capable of releasing 4MU by one enzymatic reaction without causing two enzymatic reactions.

Lead acetate or cerium acetate in the MPSII substrate solution in the above composition masks IDS enzyme reaction inhibitors such as sulfate ion, phosphate ion, citrate ion in the blood sample with lead ion or cerium ion, and the blood sample It is added to enable detection of IDS enzyme activity. However, since lead ion or cerium ion is also an interfering metal ion of Iduronidase, the addition concentration may not be too high or too low in order to properly measure the IDS enzyme activity, and an appropriate concentration setting is required. Be done. In the present invention, the lead acetate concentration or the cerium acetate concentration is set as a concentration suitable for the one-step enzymatic reaction, and when lead acetate is used, it is preferably 10 to 30 mM, more preferably 20 mM. On the other hand, when cerium acetate is used, it is preferably 2 to 20 mM, more preferably 5 mM.

The pH of the MPSII substrate solution in the above composition was set to a pH at which both IDS and IDU could react. The optimum pH for IDS is 5-6, and the optimum pH for commonly used animal-derived (rabbit) IDUs is 2-3. Therefore, it is difficult to carry out the IDS enzyme reaction and the IDU enzyme reaction at the same time. Therefore, by changing the added Iduronidase to r-IDU, which can react at pH 4 to 5, which is close to the optimum pH of IDS enzyme activity, it is possible to carry out an enzymatic reaction under the same pH conditions, and a one-step enzymatic reaction is possible. I realized the measurement of IDS enzyme activity in.

The substrate solution for detecting hypophosphatosis (hereinafter referred to as "TNSALP substrate solution") is extracted with a common extract by using a buffer solution having a buffering capacity in the pH range suitable for measuring ALP enzyme activity. It is possible to measure each enzyme using the prepared blood sample.

The synthetic substrate used in the TNSALP substrate solution needs to be a substrate having a structure that serves as a specific substrate for the enzyme to be measured. By using this synthetic substrate, the reaction specificity with the target enzyme can be improved. However, since there are innumerable enzymes and ALP isozymes that undergo similar reactions in the extracted blood sample, it is preferable to coexist with a similar enzyme inhibitor in order to further enhance the specificity.

One embodiment of the TNSALP substrate solution is "0.2 mM 4-Methylumbelliferyl phosphate (hereinafter abbreviated as" 4MU-Phos "), 0.05% TritonX-100, 10 mM sodium succinate, 10 mM L-phenylalanine (hereinafter," L- ". It is abbreviated as "Phe"), and consists of a 100 mM Tris buffer solution having a pH of 9 to 11 containing 1.0 mM MgCl ₂ .

Similar enzyme inhibitors (L-Phe) added to the TNSALP substrate solution inhibit ALP isozymes other than TNSALP (small intestinal ALP, placenta ALP, germ cell ALP) and enhance the specificity of TNSALP enzyme activity measurement. Acts on. The action may be 1 mM or more, and if it is 5 to 30 mM, the enzymatic reaction by ALP isozymes can be completely inhibited.

In the case of ALP measurement, a buffer solution of ethanolamines is generally used because an enzymatic reaction usually occurs on the alkaline side of pH 9 to 11, but the ALP concentration in the dry blood filter paper extraction sample is the serum sample. Since the activity is measured at a lower concentration than when it is used, a Tris hydroxymethane buffer solution that can be measured with higher sensitivity is preferable, and a Tris-NaOH buffer solution having a pH of 10.0 is preferable.

The substrate stabilizer (sodium succinate) added to the TNSALP substrate solution does not interfere with the action of Mg ²⁺ required for the expression of ALP activity, and promotes substrate self-decomposition such as Fe ²⁺ mixed from blood samples and other reagents. Add to inhibit divalent metal ions. The action may be 2 mM or more, preferably 5 to 30 mM.

The ALP activity enhancer (MgCl ₂ ) added to the TNSALP substrate solution supplies Mg ²⁺ necessary for the expression of ALP activity to the substrate solution. The action may be 0.1 mM or more, preferably 0.5 to 3 mM.

The common reaction product produced by each enzymatic reaction is 4MU, which emits strong fluorescence in the alkaline region, and the fluorescence intensity of this 4MU is measured. Since 4MU emits the strongest fluorescence in the alkaline range of pH 9-11, it is desirable to shift the pH of the reaction solution to the alkaline side after adding the common reaction stop solution. However, since the pH is different in each enzyme reaction and the substrate solution has a strong buffering capacity in the alkaline range, it is better to use a high-concentration alkaline buffer solution so that the common reaction terminator is not affected by them. preferable.

The common reaction terminator in the present invention is a 100 mM to 500 mM glycine-NaOH buffer (pH 9 to 11) containing a strong metal chelating agent such as ethylenediaminetetraacetic acid (EDTA). By using the common reaction terminator composition of this composition, it is possible to stop the enzymatic reaction and enhance the fluorescence of the reaction product (4MU) for measurement. In the alkaline region above pH 8, 4MU absorbs ultraviolet rays near 360 nm and emits fluorescence with maximum fluorescence intensity near 450 nm. Therefore, the pH of the common reaction terminator is preferably 8 or more, more preferably 9 or more, and even more preferably 10 to 11. The composition of the common reaction terminator that can be used in one embodiment of the present invention is most preferably "300 mM glycine-NaOH buffer (pH 10.6) containing 10 mM EDTA".

The dried blood filter paper extract contains a dye protein such as hemoglobin that interferes with 4MU fluorescence measurement, and a nucleic acid component that absorbs ultraviolet rays and inhibits 4MU excitation efficiency. In the present invention, in the measurement with a reaction solution containing a blood component, in order to measure 4MU with less inhibition of the blood component, the measurement wavelength is intentionally shifted between the maximum excitation of 4MU and the maximum fluorescence wavelength. do.

The maximum excitation wavelength of 4MU only is 360 nm, and the maximum detection wavelength is 450 nm. However, when fluorescence is measured with a blood sample mixed in, fluorescence attenuation and non-specific fluorescence generation are mixed due to the influence of blood components. It will be carried out. Therefore, in the present invention, scanning of the excitation wavelength and the detection wavelength is performed, and a combination of wavelengths that can be detected with higher sensitivity is found. This shift between the excitation wavelength and the measurement wavelength makes it possible to efficiently measure low-concentration enzyme activity, which has not been possible to measure due to the influence of blood components, and enables highly sensitive measurement.

The wavelength of the fluorescence measurement in one embodiment of the present invention is a combination of an excitation wavelength of 365 to 375 nm and a detection wavelength of 460 to 470 nm, and more preferably a combination of an excitation wavelength of 370 nm and a detection wavelength of 465 nm. By using this combination of measurement wavelengths in common for each enzyme measurement, it became possible to measure each enzyme activity even when each enzyme activity was arranged on the same microplate with one fluorescent auto reader.

Enzyme activity is calculated as the amount of 4MU produced per unit time per dry blood filter paper. The 4MU production amount is calculated by comparing the fluorescence intensity of the 4MU standard product with the fluorescence intensity of the extracted sample. The formula for calculating the enzyme activity is as follows.
Enzyme activity (pmol / hr / disk) = 4MU amount (μM) of extraction sample x extraction sample amount (μL) ÷ enzyme reaction time (hr) x (extraction solution amount per dry blood filter (μL) ÷ extraction sample Amount (μL))

In the measurement operation according to the embodiment of the present invention, one dry blood filter paper is placed in a well of a microplate, 100 to 200 μL of a common extract is added, and an extraction reaction is carried out for 1 hour. The extracted blood sample is transferred to another microplate by 10 to 20 μL by well to well, 20 to 40 μL of each substrate solution is added thereto, and the mixture is reacted at 25 to 45 ° C. for 2 to 24 hours.

The optimum enzyme reaction temperature for each enzyme reaction differs among the six responsible enzymes, and except for IDS and TNSALP, the activity is highest around 37 ° C. Therefore, the temperature condition commonly used is preferably 25 to 45 ° C, more preferably around 37 ° C.

The enzyme reaction time is generally 24 to 48 hours in the case of lysosome enzyme activity and about 5 to 30 minutes in the case of ALP enzyme activity, but the behavior differs depending on the measurement conditions, so this measurement system In the case of, the enzyme activity in 1 to 4 hours was set to be higher than that in the case of reaction for 24 hours. Therefore, 1 to 4 hours is preferable as a condition for common use. After completion of the enzymatic reaction, add 200 μL of a common reaction terminator to each reaction solution to terminate the enzymatic reaction. Using a fluorescence auto reader, fluorescence is measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, and the enzyme activity is calculated by comparing with the fluorescence intensity of the 4MU standard product.

The present invention also provides a kit for use in the inspection method of the present invention. The kit of the present invention includes the following configurations:
(1) Dry blood filter paper,
(2) Common extract,
(3) Substrate solution containing synthetic substrate,
(4) A common reaction terminator and (5) a means for measuring fluorescence of a product obtained by an enzymatic reaction.

The common extract contained in the kit of the present invention most preferably contains 0.1% BSA, 0.1% Triton X-100 and 0.05% sodium azide.

The substrate solution containing the synthetic substrate contained in the kit of the present invention is a combination of GLA substrate solution, GAA substrate solution and GBA substrate solution, MPSI substrate solution and MPSII substrate solution, and TNSALP substrate solution, and most preferably GLA substrate. The solution is "100 mM citric acid-200 mM Na phosphate buffer with pH 4.4 containing 3.0 mM 4MU-α-GAL and 100 mM GaLNAc, 0.5 mM DTT", and the GAA substrate solution is "2.0 mM 4MU-α-". A pH 4.0 250 mM citrate-250 mM potassium phosphate buffer containing Glu and 4.5 μM acarbose, 0.5 mM DTT, and the GBA substrate solution is “3.0 mM 4MU-β-Glu, 0.3% TAUDA and 0.5 mM DTT”. 100 mM citrate-100 mM Na phosphate buffer with pH 5.0 containing, and the MPSI substrate solution is "0.6 mM 4MU-IDUA, 5 μg / mL D-Sac, 0.05% Birij-35, 50 mM KCl and 0.5 mM". It is a pH 3-4 100 mM Na formic acid buffer containing DTT, and the MPSII substrate solution is a pH 4.5 50 mM acetate containing 0.13 mM 4MU-IDS, 1 μg / mL r-IDU and 20 mM lead acetate or 5 mM cerium acetate. "Na buffer", a 100 mM Tris buffer with a pH of 10.0, wherein the TNSALP substrate solution contains "0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM Na succinate, 10 mM L-Phe, 1.0 mM MgCl ₂ ,"".

The common reaction terminator contained in the kit of the present invention is most preferably a 300 mM glycine-NaOH buffer (pH 10.6) containing 10 mM EDTA.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

Specimens, reagents, etc. The specimens, reagents, etc. used in the examples are as follows.
[Samples and standard products]
・ Specimen: Dry blood filter paper (cut out to φ3 mm and used)
・ 4MU standard product: SIGMA-ALDRICH, Product No. M1381

[Synthetic substrate]
-4-MU-IDUA: Made by Toronto Research Chemical (TRC), Product No. M334701
・ 4MU-IDS: Made by Toronto Research Chemical (TRC), Product No. M334715
・ 4MU-α-GAL: SIGMA-ALDRICH, Product No. M7633
・ 4MU-α-Glu: SIGMA-ALDRICH, Product No. M9766
・ 4MU-β-Glu: SIGMA-ALDRICH, Product No. M3633
・ 4MU-Phos: SIGMA-ALDRICH, Product No. M8883

[Similar enzyme inhibitor]
・ D-Sac: SIGMA-ALDRICH, Product No. S0375
・ GaLNAc: Wako Pure Chemical Industries, Ltd., Product No. 013-1282
・ Acarbose: Wako Pure Chemical Industries, Ltd., Product No. 019-22671
・ TAUDA: SIGMA-ALDRICH, Product No. T0557P
・ TAUA: SIGMA-ALDRICH, Product No. T4009
・ L-Phe: Wako Pure Chemical Industries, Ltd., special grade reagent

[Additional enzyme]
-R-IDU (Recombinant IDU Enzyme): Made by R & D systems, Reagent Catalog Code No. 4119-GH-010

-Common extract: purified water containing 0.1% BSA, 0.1% Trtion X-100 and 0.05% sodium azide-Common reaction terminator: 10 mM EDTA, 300 mM glycine-NaOH buffer (pH 10.6)

[Measuring equipment / equipment]
・ Microplate: 96well transparent microplate (manufactured by GREINER)
-Black microplate: 96well Black microplate for fluorescence measurement (manufactured by NUNC)
・ Fluorescent auto reader: Infinite M200PRO (made by TECAN)

The "S / N ratio" is the detection sensitivity, which is a value obtained by dividing the fluorescence intensity of the obtained sample by the fluorescence intensity of each Blank sample. Further, the excitation wavelength may be described as Ex and the detection wavelength may be described as Em.

Comparison of measurement wavelength (excitation wavelength / detection wavelength) and detection sensitivity Scanning of the excitation wavelength and detection wavelength in fluorescence measurement was performed to examine the combination of wavelengths that can be detected with high sensitivity.

(1) Reagent, test solution ・ GBA substrate solution: 3.0 mM 4MU-β-Glu and 0.3% TAUDA, 0.5 mM DTT, pH 5.0 100 mM citric acid-100 mM Na phosphate buffer ・ 4MU standard solution: 4MU standard product Is dissolved in a common extract so that the final concentration is 20 μM.

(2) Experimental method
100 μL of a common extract was added to two sheets of dry blood filter paper, and the mixture was extracted for 2 hours. Add 10 μL of the extraction sample (10 μL of the common extract only in the case of Blank), 10 μL of the 20 μM 4MU standard solution, 40 μL of the GBA substrate solution, and 200 μL of the common reaction stop solution to the wells of the microplate, and (i) set the detection wavelength to 450 nm. It was fixed at 460 nm and the excitation wavelengths of 300 nm to 400 nm were scanned every 5 nm. In addition, (ii) the excitation wavelengths were fixed at 360 nm and 365 nm, and the detection wavelengths of 400 nm to 500 nm were scanned every 5 nm. After that, the S / N ratio was calculated and the detection sensitivities were compared.

(3) Results As shown in FIG. 1, a higher S / N ratio was obtained at an excitation wavelength of 370 to 380 nm and a detection wavelength of 460 nm, which is a longer wavelength than 450 nm. Further, as shown in FIG. 2, a high S / N ratio was obtained at an excitation wavelength of 365 nm on the long wavelength side of 360 nm and a detection wavelength of 460 to 480 nm on the long wavelength side of 450 nm. When these results were combined, it was found that the sample containing the blood component had the highest S / N ratio at the excitation wavelength of 370 to 380 nm and the detection wavelength of 460 to 480 nm.

Confirmation of higher sensitivity by measuring wavelength shift Whether or not the wavelength shift according to Example 1 is effective even when measuring an actual sample is longer than when measured under general conditions (Ex365 / Em450, Ex360 / Em460). The comparison was made with the case of measurement with a combination of wavelength shifts to the wavelength side.

(1) Reagent / Test solution / Substrate solution:
(i) GLA substrate solution: 100 mM citric acid-200 mM Na phosphate buffer with pH 4.4 containing 3.0 mM 4MU-α-GAL and 100 mM GaLNAc, 0.5 mM DTT
(ii) GAA substrate solution: 250 mM citric acid-250 mM potassium phosphate buffer at pH 4.0 containing 2.0 mM 4MU-α-Glu and 4.5 μM acarbose, 0.5 mM DTT
(iii) GBA substrate solution: 100 mM citric acid-100 mM Na phosphate buffer at pH 5.0 containing 3.0 mM 4MU-β-Glu and 0.3% TAUDA, 0.5 mM DTT

(2) Experimental method A dried blood filter paper sample was cut into each well of a microplate, 100 μL of a common extract was added to each well, and an extraction operation was performed for 1 hour while shaking at room temperature. 20 μL of the extracted blood sample was transferred to a Black microplate by well to well. 40 μL of each substrate solution was added to the well containing the sample, and the mixture was allowed to stand in a constant temperature bath at 37 ° C. for 4 hours to carry out an enzymatic reaction. After the enzyme reaction is completed, 200 μL of the common reaction terminator is added to each well, and after lightly mixing, the fluorescence intensity is measured for each combination of excitation wavelength and detection wavelength using a fluorescence auto reader, and the S / N ratio is calculated. did.

(3) Results Figures 3 to 5 show the S / N ratios of dried blood filter paper (N = 3 / Samples 1 to 3) when GLA, GAA, and GBA are measured with each combination of measurement wavelengths. For GLA, GAA, and GBA, the combination of wavelengths shifted to the longer wavelength side shows a higher S / N ratio than when measured at the excitation wavelength of 365 nm and the detection wavelength of 450 nm, which are the general conditions, and the sample is displayed with high sensitivity. Measurements using actual dry blood filter paper also showed that it could be measured.

Verification of the effect of adding acarbose on GAA substrate solution
The effect of adding acarbose on the GAA substrate solution was compared using the dry blood filter paper of Pompe patients and the dry blood filter paper of normal humans (normal newborns).

(1) Reagents-Test solution-GAA substrate solution: 2.0 mM 4MU-α-Glu-containing 250 mM citric acid-250 mM potassium phosphate buffer solution Two types of the above substrate solution, with and without addition of 4.5 μM acarbose. Made.
(2) Experimental method Example 2 "(2) Experimental method" was used as a dry blood filter paper collected from a normal person (newborn) (N = 74) and from a Pompe patient (N = 1). From extraction to GAA enzyme reaction was carried out in the same manner as above. After that, the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, and the GAA enzyme activity per dry blood filter paper was determined by comparing with the fluorescence intensity of the 4MU standard solution.

(3) Results The results are shown in FIG. It was shown that the use of a substrate solution supplemented with acarbose at 4.5 μM suppresses substrate degradation by α-glucosidase other than the GAA enzyme, making it possible to distinguish between patients and normal individuals.

Comparison of additives (TAUA and TAUDA) in GBA enzyme activity measurement
We investigated whether changing the additive of GBA substrate solution from TAUA to TAUDA would be effective in enhancing the measurement of GBA enzyme activity.

(1) Reagents / Test solutions- (i) GBA substrate solution: 3.0 mM 4MU-β-Glu, 0.5 mM DTT-containing 100 mM citric acid-100 mM Na phosphate buffer with pH 5.0 In the above substrate solution, (ii) 0.2 Three types were prepared: one to which% TAUDA was added and one to which (iii) 0.2% TAUA was added.

(2) Experimental method The process from extraction to GBA enzyme reaction was carried out in the same manner as in Example 2 “(2) Experimental method”. Then, 200 μL of the common reaction terminator was added to each well, and after light mixing, the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, the S / N ratio was calculated, and the detection sensitivities were compared.

(3) Results The results are shown in Table 1. It was found that in all of Samples 1 to 3, the S / N ratio was higher when TAUDA was used than when TAUA was used, and it was possible to measure with higher sensitivity.

Enzyme activity enhancing effect with or without DTT
The GLA, GAA, and GBA enzyme activities of the sample extracted from the dry blood filter paper were simultaneously measured using the substrate solution containing DTT and the substrate solution not containing DTT, and the enzyme activity enhancing effect of DTT was confirmed.

(1) Reagent / Test solution / Substrate solution
(i) GLA substrate solution: 100 mM citric acid-200 mM Na phosphate buffer with pH 4.4 containing 3.0 mM 4MU-α-GAL, 100 mM GaLNAc
(ii) GAA substrate solution: 250 mM citric acid-250 mM potassium phosphate buffer with pH 4.0 containing 2.0 mM 4MU-α-Glu and 4.5 μM acarbose.
(iii) GBA substrate solution: 3.0 mM 4MU-β-Glu, pH 5.0 100 mM citric acid-100 mM Na phosphate buffer containing 0.3% TAUDA
For each of (i) to (iii), two types of substrate solutions with and without 0.5 mM DTT were prepared.

(2) Experimental method The enzyme reaction was carried out by the same method as described in Example 2 "(2) Experimental method" except that the extraction was carried out by allowing the mixture to stand at room temperature for 1 hour and shaking for 1 hour. .. After that, 200 μL of the common reaction terminator was added to each well, and after light mixing, the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, and the fluorescence intensity of the sample measured with the substrate solution containing no DTT was set to 100%. The ratio (%) of the fluorescence intensity of the sample measured with the substrate solution containing DTT was compared.

(3) Results The results are shown in FIG. It was confirmed that the DTT concentration of 0.5 mM did not affect the enzyme activity of GLA and GAA, and only GBA was enhanced.

Enhancement of blood non-specific fluorescence with or without DTT addition By adding DTT to the substrate solution, GBA enzyme activity is enhanced, but when DTT becomes high concentration, blood components in the sample react with DTT to enhance specific fluorescence. .. In order to verify this phenomenon, a substrate solution containing no 4MU synthetic substrate (hereinafter referred to as "substrate blank solution") was prepared, and it was confirmed to what extent the enhancement of non-specific fluorescence occurs depending on the presence or absence of DTT addition.

(1) Reagent / Test solution / Substrate blank solution: 100 mM citric acid-100 mM Na phosphate buffer with pH 5.0 containing 0.3% TAUDA Two types of the above substrate blank solution were prepared with and without 5 mM DTT.

(2) Experimental method Example 2 "(2) Experiment" except that the extract was allowed to stand at room temperature for 1 hour and shaken for 1 hour, and the extracted blood sample was transferred to two microplates. The process from the enzymatic reaction to the transfer was carried out by the same method as described in "Method". Then, 40 μL of each substrate blank solution (with and without DTT) was added to the well containing the sample, and the mixture was allowed to stand in a constant temperature bath at 37 ° C. for 4 hours to carry out the reaction. After completion of the reaction, 200 μL of the common reaction terminator was added to each well, and after light mixing, the fluorescence intensities were measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, and the fluorescence intensities of the substrate blank solution (with / without DTT) were compared. ..

(3) Results The results are shown in FIG. It was confirmed that in all of Samples 1 to 4, when the substrate blank solution to which DTT was added at a concentration of 5 mM was used, the components of the blood extract and DTT reacted and the non-specific fluorescence was enhanced. Therefore, it was found that the DTT concentration of the substrate solution should be kept low.

Comparison with the method described in the prior art document When the measurement method using the individual enzyme measurement method described in the prior art document (Non-Patent Documents 1 and 2) and the measurement method according to the present invention are measured using the same dried filter paper blood. The measurement sensitivities of were compared by the S / N ratio of the fluorescence intensity.

[Measurement method described in the prior art document]
(i) GLA: Measured according to the measurement conditions described in Non-Patent Document 1. Add 40 μL of extract (McIlvan Buffer) to one dry blood filter paper and extract for 2 hours. After mixing 30 μL of the extracted sample and 100 μL of the substrate solution and reacting at 37 ° C. for 24 hours, 150 μL of the reaction stop solution was added, and the 4MU fluorescence intensity was measured at Ex365 nm and Em450 nm.
(ii) GAA: Measured according to the measurement conditions described in Non-Patent Document 2. Add 100 μL of extract (ion-exchanged water) to one dry blood filter paper and extract for 2 hours. After mixing 20 μL of the extracted sample and 40 μL of the substrate solution and reacting at 37 ° C. for 24 hours, 190 μL of the reaction stop solution was added, and the 4MU fluorescence intensity was measured at Ex360 nm and Em 450 nm.
(iii) GBA: Measurement by partially reorganizing the measurement conditions described in Non-Patent Document 1. Add 40 μL of extract (McIlvan Buffer) to one dry blood filter paper and extract for 2 hours. After mixing 30 μL of the extracted sample and 100 μL of the substrate solution and reacting at 37 ° C. for 24 hours, 150 μL of the reaction stop solution was added, and the 4MU fluorescence intensity was measured at Ex365 nm and Em450 nm.
* Substrate solution: 100 mM citric acid-phosphate buffer of ph5.0 containing 10 mM 4MU-β-Glu and 0.2% TAUA

[Measurement method according to the present invention]
(1) Reagent / Test solution / Substrate solution: GLA substrate solution, GAA substrate solution, GBA substrate solution, all of which have the composition described in Example 2 “(1) Reagent / Test solution” are used.

(2) Measurement method Extraction to enzymatic reaction were carried out in the same manner as in Example 2 “(2) Measurement method”. Then, 200 μL of the common reaction terminator was added to each well, and after light mixing, the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, and the S / N ratio was calculated and compared under each condition.

(3) Results The results are shown in FIG. As a result of measurement using the same dry blood filter paper as a sample by the measurement method described in the prior art document and the measurement method according to the present invention, the measurement method according to the present invention has an S / N ratio for all three measurement items. It became higher, and it was shown that the measurement can be performed with higher sensitivity and in a shorter time than the measurement conditions described in the prior art literature.

Confirmation of enhancement of IDU enzyme activity
In order to enhance the IDU enzyme activity, various additives were added to the substrate solution for measurement.
(1) Specimens, reagents, etc.-Samples: r-IDU
-MPSI substrate solution: pH 3.5 100 mM Na formic acid buffer containing 0.6 mM 4MU-IDUA

(2) Measurement method
r-IDU was adjusted to 8 ng / mL with a common extract, and 20 μL of this diluted standard enzyme was transferred to a Black microplate as a sample. 20 μL of the substrate solution containing each additive was added to the well containing the sample, and the mixture was allowed to stand in a constant temperature bath at 37 ° C. for 4 hours to carry out an enzymatic reaction. After completion of the enzymatic reaction, 200 μL of the common reaction terminator was added to each well and mixed lightly, and then the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm.

(3) Results The results are shown in FIG. TAUDA, which has an enhancing effect on GBA, and lead acetate, which is essential for IDS measurement, inhibited the IDU enzyme activity. Addition of Birji-35 and KCl showed enhanced IDU enzyme activity.

Examination of KCl addition concentration for enhancing IDU enzyme activity
The concentration of KCl added to the substrate solution was investigated to enhance the IDU enzyme activity.

(1) Specimens, reagents, etc.-Samples: r-IDU
MPSI substrate solution: pH 3.5 100 mM Na buffer solution containing 0.6 mM 4MU-IDUA, 5 μg / mL D-Sac, 0.05% Birij-35 and 0.25 mM DTT For the above substrate solution, (i) without KCl, ( Four types were prepared: ii) addition of 30 mM KCl, (iii) addition of 60 mM KCl, and (iv) addition of 120 mM KCl.

(2) Measurement method Measurement was performed by the same method as described in Example 8 “(2) Measurement method”.

(3) Results The results are shown in FIG. By adding KCl in the range of 30 mM to 120 mM, an enzyme activity enhancing effect of 30% or more was shown as compared with the case of no addition.

Comparison of acetic acid buffer and formic acid buffer
The optimum substrate buffer for measuring IDU enzyme activity was compared between acetate buffer (pH 3.0) and formic acid buffer (pH 3.0).
(1) Specimens, reagents, etc.-Samples: Normal human dry blood filter paper (dry blood filter paper for 74 newborns)
・ MPSI substrate solution:
(i) pH 3.0 100 mM Na Formic Acid Buffer containing 0.6 mM 4MU-IDUA, 5 μg / mL D-Sac, 0.05% Birij-35, 50 mM KCl
(ii) pH 3.0 100 mM Na acetate buffer containing 0.6 mM 4MU-IDUA, 5 μg / mL D-Sac, 0.05% Birij-35, 50 mM KCl

(2) Measurement method A dried blood filter paper sample was cut into each well of a microplate, 200 μL of a common extract was added to each well, and an extraction operation was performed for 1 hour while shaking at room temperature. The extracted blood samples were transferred well to well into two Black microplates of 20 μL each. For the 4MU standard product, 20 μL of the diluted standard solution diluted 2-fold from 2.5 μM was transferred to the Black microplate in the same manner as the blood sample. 20 μL of each synthetic substrate was added to the wells containing the sample and the 4MU standard solution, and the mixture was allowed to stand in a constant temperature bath at 37 ° C. for 4 hours for enzymatic reaction. After the enzymatic reaction is completed, 200 μL of the common reaction terminator is added to each well, and after light mixing, the fluorescence intensity is measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, and each dry blood filter paper is compared with the fluorescence intensity of the 4MU standard solution. The enzyme activity per sheet was determined.

(3) Results The results are shown in FIG. Both are pH 3.0 buffers, but it was confirmed that the use of formic acid buffer resulted in higher enzyme activity measurement results. This is considered to be due to the reducing action of the formic acid buffer solution.

Examination of extract buffer (IDS enzyme activity measurement)
The IDS enzyme activity was compared using a phosphate-citrate buffer and a 0.1% BSA-H ₂ O buffer as the extract buffer.
(1) Specimens, reagents, etc.-Samples: Genetically modified Iduronate 2-sulfatase (hereinafter abbreviated as "r-IDS")
Reagent Catalog Code No. 2449-SU-020r-IDS, manufactured by R & D systems
・ Extract:
(i) Purified water containing 0.1% BSA, 0.1% Trtion X-100 and 0.05% sodium azide
(ii) 25 mM citric acid-25 mM potassium phosphate buffer / MPSII substrate solution at pH 6.0 containing 0.1% TrtionX-100, 0.05% sodium azide: 0.13 mM 4MU-IDS, 1 μg / mL r-IDU and 5 mM acetic acid 50 mM Na acetate buffer with pH 4.5 containing cerium

(2) Measurement method
The r-IDS was adjusted to 125 to 1000 ng / mL with each extract, and 20 μL of this diluted standard enzyme was transferred to a Black microplate as a sample. 20 μL of the substrate solution containing each additive was added to the well containing the sample, and the mixture was allowed to stand in a constant temperature bath at 37 ° C. for 4 hours to carry out an enzymatic reaction. After completion of the enzymatic reaction, 200 μL of the common reaction terminator was added to each well and mixed lightly, and then the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm.

(3) Results The results are shown in FIG. IDS enzyme activity could not be measured when phosphate-citrate buffer was used.

Examination of optimum pH of MPSII substrate solution
The optimum pH was examined by measuring by changing the pH of the MPSII substrate solution.
(1) Specimens, reagents, etc.-Samples: r-IDS
-Substrate solution: 50 mM Na acetate buffer containing 0.13 mM 4MU-IDS, 1 μg / mL r-IDU and 5 mM cerium acetate For the above substrate solution, (i) pH4.0, (ii) pH4.5, (iii) pH5 Three types of .0 were prepared.

(2) Measurement method
Except that r-IDS was adjusted to 500 ng / mL with a common extract, the enzyme reaction to the fluorescence intensity measurement were carried out in the same manner as in the method described in Example 11 (2) Measurement Method.

(3) Results The results are shown in FIG. Since the enzyme activity was maximized at around pH 4.5, it was found that the optimum pH of the MPSII substrate solution was around 4.5.

Examination of the effect of adding cerium acetate Acetic acid as an additive for masking IDS enzyme reaction inhibitors such as sulfate ion, phosphate ion, and citrate ion in the blood sample in order to measure the IDS enzyme activity in the blood extraction sample. Add lead or cerium acetate to the substrate solution. Generally, lead acetate is often used, but the effect of adding cerium acetate as another additive was investigated in order to eliminate the complexity of waste liquid treatment after measurement.
(1) Specimens, reagents, etc.-Samples: Normal human dry blood filter paper-MPSII substrate solution:
(i) pH 4.5 50 mM Na acetate buffer containing 0.13 mM 4MU-IDS, 1 μg / mL r-IDU
(ii) 50 mM Na acetate buffer with pH 4.5 containing 0.13 mM 4MU-IDS, 1 μg / mL r-IDU and cerium acetate (prepared with cerium acetate concentrations of 5 mM, 10 mM, 20 mM and 30 mM)
(iii) pH 4.5 50 mM Na acetate buffer containing 0.13 mM 4MU-IDS, 1 μg / mL r-IDU and 20 mM lead acetate

(2) Measurement method The process from extraction to enzymatic reaction was carried out in the same manner as in Example 2 “(2) Experimental method”. Then, 200 μL of the common reaction terminator was added to each well, and after light mixing, the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, the S / N ratio was calculated, and the detection sensitivities were compared.

(3) Results The results are shown in FIG. It was found that cerium acetate can be used at a lower concentration than lead acetate. It can be said that cerium acetate is not a heavy metal compound and is an effective additive because it facilitates waste liquid treatment after the measurement is completed.

Effect of adding metal chelating agents (EDTA, citric acid, succinic acid) to the substrate solution
The coexistence of divalent metal ions such as Mg ²⁺ ions is required for the expression of the enzyme activity of ALP, but on the other hand, a large amount of divalent metal ions such as Fe ²⁺ derived from hemoglobin are present in the whole blood. The presence of a large amount of divalent metal ions other than Mg ²⁺ promotes self-decomposition of the synthetic substrate, so a metal chelating agent (EDTA, citric acid, succinic acid) that does not inhibit the effect of Mg ²⁺ ions can be added to the substrate solution. The effect of addition was investigated.

(1) Specimens, reagents, etc.-Samples: Normal human dry blood filter paper-TNSALP substrate solution: (i) pH 10.0 containing 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM L-Phe, 1.0 mM MgCl ₂ . 100 mM Tris buffer solution Based on the above substrate solution, a substrate solution containing (ii) 4 mM EDTA · 4Na, (iii) 4 mM Na citrate, and (iv) 4 mM Na succinate was also prepared.

(2) Measurement method Extraction, enzyme reaction, and fluorescence intensity were measured by the same method as in Example 13 “(2) Measurement method”.

(3) Results The results are shown in FIG. For EDTA (chelate constant 16.5), citric acid (chelate constant 11), and succinic acid (chelate constant 13), it was found that the addition of citric acid or succinic acid, which has a weak chelating effect, does not inhibit ALP measurement.

Examination of addition concentration of Na citrate and Na succinate Using dried blood filter paper as a sample, TNASAPL enzyme activity was measured by changing the addition concentration of Na citrate or Na succinate in the substrate solution.

(1) Specimens, reagents, etc.-Samples: Normal human dry blood filter paper-TMSALP substrate solution: 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM L-Phe, 1.0 mM MgCl ₂ , pH 10.0 100 mM Tris Buffer solution Based on the above substrate solution, a substrate solution containing (i) 0 to 8 mM Na citrate and (ii) 0 to 8 mM Na succinate was prepared.

(2) Measurement method Extraction, enzymatic reaction and measurement were carried out by the same method as described in Example 13 (2) Measurement method.

(3) Results The results are shown in FIG. It was found that citric acid decreased the enzyme activity at 4 mM or more, but succinic acid did not affect the enzyme activity measurement.

Examination of L-Phe concentration in TNSALP substrate solution
The optimum concentration of L-Phe to be added to the TNSALP substrate solution was investigated.

(1) Specimens, reagents, etc.-Samples: Normal human dry blood filter paper (samples 1 to 3)
TNSALP substrate solution: pH 10.0 100 mM Tris buffer containing 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM Na succinate, 0-8 mM L-Phe, 1.0 mM MgCl ₂ .

(2) Measurement method Extraction, enzymatic reaction and measurement were carried out by the same method as described in Example 13 “(2) Measurement method”.

(3) Results The results are shown in FIG. L-Phe is an inhibitor of ALP, but its sensitivity to each isozyme is different. The 50% inhibitory concentration of TNSALP is 31 mM, the small intestine type is 0.8 mM, the placental type is 1.1 mM, and the germ cell type is 0.8 mM. In this measurement, it is desirable to set the concentration within the range of a concentration lower than the 50% inhibitory concentration of TNSALP and more than twice the 50% inhibitory concentration of ALP other than TNSALP. Therefore, it was found that ALP that can be measured by this measurement system can reflect the enzymatic activity of TNSALP when used at a concentration of 2 mM or more.

Simultaneous measurement of 6 types (enzyme activity)
Using dried blood filter paper as a sample, the enzyme reaction time was changed from 2 to 24 hours, and the activity of 6 kinds of enzymes was measured at the same time.
(1) Specimens, reagents, etc.-Normal human dry blood filter paper (dry blood filter paper for 74 newborns)
-GLA substrate solution, GAA substrate solution, GBA substrate solution: Use the one having the composition described in Example 2 "(1) Reagent / Test Solution" -MPSI substrate solution: 0.6 mM 4MU-IDUA, 5 μg / mL D-Sac, 100 mM Na formic acid buffer at pH 3.5 containing 0.05% Birij-35, 50 mM KCl and 0.25 mM DTT ・ MPSII substrate solution: 0.13 mM 4MU-IDS, 1 μg / mL r-IDU and pH 4.5 containing 5 mM cerium acetate 50 mM Na acetate buffer ・ TNSALP substrate solution: pH 10.0 100 mM Tris buffer containing 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM Na succinate, 10 mM L-Phe, 1.0 mM MgCl ₂ ,

(2) Measurement operation A dried blood filter paper sample was cut into each well of a microplate, 150 μL of a common extract was added to each well, and an extraction operation was performed for 1 hour while shaking at room temperature. The extracted blood samples were transferred well to well into 6 Black microplates of 20 μL each. For 4MU standard products, except for TNSALP, a diluted standard solution diluted 2-fold from 2.5 μM, and in the case of TNSALP, diluted 2-fold serially diluted 20 μM was transferred to a Black microplate in the same manner as the blood sample. 30 μL of each synthetic substrate was added to the wells containing the sample and the 4MU standard solution, and the mixture was allowed to stand in a constant temperature bath at 37 ° C. for 2 to 24 hours for the enzymatic reaction. After the enzymatic reaction is completed, 200 μL of the common reaction terminator is added to each well, and after light mixing, the fluorescence intensity is measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm, and each dry blood filter paper is compared with the fluorescence intensity of the 4MU standard solution. The enzyme activity per sheet was determined.

(3) Results The results are shown in FIGS. 19 and 20. The behavior differs depending on each enzyme, but the activity value of many enzymes decreases after a 24-hour reaction. Therefore, it was found that the reaction time when used in this measurement method should be set within 4 hours.

Simultaneous measurement of 6 types (normal human sample)
Using dry blood filter paper as a sample, the activity of 6 kinds of enzymes was measured at the same time. As a sample, a normal human dry blood filter paper (dry blood filter paper for 1000 newborn babies) was used. The method is the same as the method shown in Example 17 except that the enzyme reaction time is set to 3 hours.

Simultaneous measurement of 6 types (patient sample)
Dry blood filter paper was used as a sample, and dry blood filter paper of Fabry disease patient, Pompe disease patient, Gauche disease patient, MPSI disease patient, MPSII disease patient, and hypophosphatase disease patient was measured as a sample. The method is the same as the method shown in Example 18.

The specimens used in Examples 18 and 19 were 1157 normal subjects (newborn dry blood filter paper), 1 Fabry disease patient, 1 Pompe disease patient, 1 Gauche disease patient, 1 MPSI disease patient, and MPSII disease. One patient and one patient with hypophosphatase disease.

The distribution of each enzyme activity in normal people and the enzyme activity in patients with each disease are shown in FIGS. 22 to 27 and Table 2. The enzyme activity of patients with each disease was clearly lower than that of normal people.

Therefore, the measurement of the present invention showed that a rapid mass screening test for 6 diseases is possible by simultaneously measuring the activity of 6 kinds of enzymes using one sheet of dry blood filter paper.

The measuring method according to the present invention can be used in newborn mass screening tests and the like.

Claims (42)

A rapid mass screening test method for inborn errors of metabolism that rapidly measures the activity of the responsible enzyme for multiple inborn errors of metabolism from a single sheet of dry blood filter paper.
(Group A) α-D-galactosidase A (GLA), acidic α-glucosidase (GAA), acidic β-glucosidase (GBA)
(Group B) α-L-Iduronidase (IDU), Iduronate-2-sulfatase (IDS) and (Group C) Tissue non-specific alkaline phosphatase (TNSALP)
Enzymes of 1 or more in each group selected from at least two groups of :
(1) A step of extracting a blood sample from a dry blood filter paper using a common extract.
(2) A step of adding a substrate solution containing a synthetic substrate to the extracted blood sample for each responsible enzyme to carry out an enzymatic reaction.
(3) Includes a step of measuring fluorescence of the product obtained by the enzymatic reaction.
Here, the common extract comprises bovine serum albumin (BSA), Triton X-100 and sodium azide.
Method. The method of claim 1, wherein the common extract comprises 0.02% to 0.1% sodium azide , 0.05% to 0.5% Triton X-100 and 0.05% to 0.5% BSA. If the substrate solution is the responsible enzyme of Group A, (i) a buffer solution containing N-acetylgalactosamine and a 4-Methylumbelliferyl synthetic substrate, (ii) a buffer solution containing acarbose and a 4-Methylumbelliferyl synthetic substrate, and (iii). ) A combination of buffers containing 4-Methylumbelliferyl synthetic substrate, in the case of Group B responsible enzymes, (iv) buffers containing potassium chloride and 4-Methylumbelliferyl synthetic substrate, and (v) Iduronidase and 4-Methylumbelliferyl synthesis. The method according to claim 1 or 2 , which is a combination of a buffer containing a substrate, and in the case of the responsible enzyme of Group C, (vi) a buffer containing L-phenylalanine and a 4-Methylumbelliferyl synthetic substrate. The method according to claim 3 , wherein the 4-Methyum belliferyl synthetic substrate in (i) is 4-methylum-belliferyl-α-D-galactopyranoside and is contained in an amount of 1 mM to 10 mM. The method according to claim 3 or 4 , wherein the pH of the substrate solution in (i) is 4.0 to 5.0. The method according to any one of claims 3 to 5 , wherein the substrate solution in (i) is a citric acid-sodium phosphate buffer solution containing 0.1 mM to 1.0 mM Dithiothreitol (DTT). The method according to claim 3 , wherein the 4-Methyum belliferyl synthetic substrate in (ii) is 4-methylum-belliferyl-α-D-glucopyranoside, which is contained in an amount of 1 mM to 10 mM and acarbose in an amount of 1 μM to 10 μM. The method according to claim 3 or 7 , wherein the pH of the substrate solution in (ii) is 3.5 to 4.5. The method according to claim 3 , 7 or 8 , wherein the substrate solution in (ii) is a citric acid-potassium phosphate buffer solution containing 0.1 mM to 1.0 mM Dithiothreitol (DTT). The method according to claim 3 , wherein the 4-Methyum belliferyl synthetic substrate in (iii) is 4-methylum-belliferyl-β-D-glucopyranoside, which is contained in an amount of 1 mM to 10 mM. The method according to claim 3 or 10 , wherein the pH of the substrate solution in (iii) is 4.5 to 5.5. The method according to claim 3 , 10 or 11 , wherein the substrate solution in (iii) is a citric acid-sodium phosphate buffer solution. The method according to any one of claims 3 and 10 to 12 , wherein the substrate solution in (iii) contains sodium taurodeoxycholate as a similar enzyme inhibitor. The method according to any one of claims 3 and 10 to 13 , wherein the substrate solution in (iii) contains 0.1 mM to 1 mM Dithiothreitol (DTT). The method according to claim 3 , wherein the 4-Methyumbelliferyl synthetic substrate in (iv) is 4-Methylumbelliferyl α-L-Idopyranosiduronic Acid, Sodium Salt, which is contained in an amount of 0.1 mM to 3 mM. The method according to claim 3 or 15 , wherein the pH of the substrate solution in (iv) is 3 to 4. The method according to claim 3 , 15 or 16 , wherein the buffer solution of the substrate solution in (iv) has a reducing action. 17. The method of claim 17 , wherein the buffer is a sodium formate buffer. The method according to any one of claims 3 and 15 to 18 , wherein the substrate solution in (iv) contains 0.1 mM to 1 mM Dithiothreitol (DTT). The method according to any one of claims 3 and 15 to 19 , wherein the substrate liquid in (iv) contains 0.01% to 0.1% of Polyoxyethyleneglycol Dodecyl Ether (Brij-35). The invention according to any one of claims 3 and 15 to 20 , wherein the substrate solution in (iv) contains 1 μg / mL to 20 μg / mL of D-Saccharic acid 1,4-lactone as a similar enzyme inhibitor. Method. The method according to claim 3 , wherein the Iduronidase in (v) is an Iduronidase produced by a gene recombination technique and is contained at 0.1 μg / mL to 2 μg / mL. The method according to claim 3 or 22 , wherein the 4-methylumbelliferyl synthetic substrate in (v) is 4-Methylumbelliferyl-aL-Idopyranosiduronic Acid-2-Sulfate, Sodium Salt, which is contained in an amount of 0.1 mM to 3 mM. The method according to claim 3 , 22 or 23 , wherein the pH of the substrate solution in (v) is 4 to 5. The method according to any one of claims 3 and 22 to 24 , wherein the buffer solution of the substrate solution in (v) is a sodium acetate buffer solution. The method according to any one of claims 3 and 22 to 25 , wherein the substrate liquid in (v) contains 10 mM to 30 mM lead acetate or 2 to 20 mM cerium acetate. The method according to claim 3 , wherein the 4-methylumbelliferyl synthetic substrate in (vi) is 4-Methylumbelliferylphosphate, which is contained in an amount of 0.05 mM to 2 mM. The method according to claim 3 or 27 , wherein the pH of the substrate solution in (vi) is 9 to 11. The method according to claim 3 , 27 or 28 , wherein the buffer solution of the substrate solution in (vi) is a Tris buffer solution. The method according to any one of claims 3 and 27 to 29 , wherein the substrate liquid in (vi) contains 1 mM to 30 mM L-phenylalanine. The method according to any one of claims 3 and 27 to 30 , wherein the substrate solution in (vi) contains 0.5 mM to 2 mM MgCl ₂ as an enzyme activity enhancer. The method according to any one of claims 3 and 27 to 31 , wherein 1 mM to 30 mM sodium succinate is contained as a substrate autolysis inhibitor in the substrate liquid in (vi). The method according to any one of claims 1 to 32 , wherein in the step (2), the enzyme reaction is stopped by adding a common reaction stop solution containing glycine and EDTA to the enzyme reaction solution. 33. The method of claim 33 , wherein the common reaction terminator has a pH of 10-11. The method according to claim 33 or 34 , wherein the enzymatic reaction is carried out at a temperature of 25 to 45 ° C., and then the common reaction terminator is added. The method according to any one of claims 1 to 35 , wherein the enzymatic reaction is carried out in 1 to 4 hours. The method according to any one of claims 1 to 36 , wherein the measurement wavelength of the fluorescence measurement is an excitation wavelength of 365 to 375 nm and a detection wavelength of 460 to 470 nm. A method for simultaneously extracting multiple enzymes responsible for inborn errors of metabolism from a single sheet of dry blood filter paper, including the use of a common extract.
(Group A) α-D-galactosidase A (GLA), acidic α-glucosidase (GAA), acidic β-glucosidase (GBA)
(Group B) α-L-Iduronidase (IDU), Iduronate-2-sulfatase (IDS) and (Group C) Tissue non-specific alkaline phosphatase (TNSALP)
Enzymes of one or more in each group, selected from at least two groups of
Here, the method, wherein the common extract comprises bovine serum albumin (BSA), Triton X-100 and sodium azide . 38. The method of claim 38 , wherein the common extract comprises 0.02% to 0.1% sodium azide , 0.05% to 0.5% Triton X-100 and 0.05% to 0.5% B SA. A kit for use in the inspection method of claim 1, including:
(1) Dry blood filter paper,
(2) Common extract,
(3) Substrate solution containing synthetic substrate,
(4) Common reaction terminator solution, and (5) Means for measuring fluorescence of the product obtained by the enzymatic reaction.
Here, the common extract contains 0.1% BSA, 0.1% Trtion X-100 and 0.05% sodium azide. The substrate solution containing the synthetic substrate is a substrate solution for measuring GLA enzyme activity for detecting Fabry's disease, a substrate solution for measuring GAA enzyme activity for detecting Pompe's disease, and a GBA enzyme for detecting Gauche's disease. Substrate solution for activity measurement, IDU enzyme substrate for detecting mucopolysaccharidosis type I (MPSI) MPSI substrate solution for measuring mucopolysaccharidosis II (MPSII), MPSII substrate for detecting mucopolysaccharidosis II (MPSII) In combination with the solution and the TNSALP substrate solution for detecting hypophosphatase, the substrate solution for measuring GLA enzyme activity for detecting Fabry's disease is 3.0 mM 4-methylumbelliferyl-α-D-garactopyranoside and 100 mM N. -Acetyl-D-galactosamine, pH 4.4 100 mM citrate containing 0.5 mM DTT-200 mM sodium phosphate buffer; 2.0 mM 4-methylumbelliferyl- is a substrate solution for measuring GAA enzyme activity to detect Pompe disease. 250 mM citrate-250 mM potassium phosphate buffer at pH 4.0 containing α-D-glucopyranoside and 4.5 μM acarbose, 0.5 mM DTT; 3.0 mM 4 substrate solution for GBA enzyme activity measurement to detect Gauche disease -Methylumbelliferyl-β-D-glucopyranoside, 0.5 mM DTT and 0.3% sodium taurodeoxycholate, pH 5.0 100 mM citrate-100 mM sodium phosphate buffer; MPSI substrate solution is 0.6 mM 4-methylumbelliferyl-α- 100 mM sodium formate buffer at pH 3-4 containing L-Idopyranosiduronic acid, sodium salt, 5 μg / mL D-Saccharic acid 1,4-lactone, 0.05% Birij-35, 50 mM KCl and 0.25 mM DTT; MPSII substrate solution, 0.13 mM 4-methylumbelliferyl-α-L-Idopyranosiduronic Acid -2-sulfate, Sodium Salt (4MU-IDS), 1 μg / mL recombinant Iduronidase and 20 mM lead acetate or 50 mM pH 4.5 containing 2-20 mM cerium acetate Sodium acetate buffer; TNSALP substrate solution is 0.2 mM The kit according to claim 40 , which is a 100 mM Tris buffer of pH 9-11 containing 4-methylumbelliferyl-phosphate, 0.05% TritonX-100, 10 mM sodium succinate, 10 mM L-phenylalanine, 1 mM MgCl ₂ . The kit according to claim 40 or 41 , wherein the common reaction terminator is a 300 mM glycine-NaOH buffer (pH 10.6) containing 10 mM EDTA.

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