JP2018102294A - Mass screening test method for hypophosphatasia using dry blood filter paper - Google Patents

Abstract

In a newborn mass screening test, a sample extracted from a dry blood filter paper is used as a measurement specimen. The conventional metabolic abnormality test cannot perform a screening test for hypophosphatasia, and the conventional measurement of alkaline phosphatase activity by a biochemical test method cannot measure using a dry blood filter paper as a specimen. Provided is a method for practically simple, quick and inexpensive implementation of hypophosphatasia responsible enzyme activity using dry blood filter paper without requiring a large-scale measuring instrument or dedicated device. SOLUTION: Provided is a method for measuring low phosphatase responsible enzyme activity using an inexpensive substrate solution with a reaction time suitable for mass screening test and simple operation using a sample extracted from a sheet of dry blood filter paper Thus, a method for detecting hypophosphatasia is provided. [Selection figure] None

Description

  The present invention relates to a simple screening test for hypophosphatasia using dry blood filter paper.

  Hypophosphatasia is one of the bone system diseases caused by deficiency of tissue non-specific alkaline phosphatase (hereinafter referred to as “TNSALP”). Based on the onset and spread of symptoms, "perinatal type" that develops in the womb, "infant type" that develops in the second half of life, "pediatric type" that develops in childhood with early loss of the deciduous teeth, adulthood It is classified into five types, "Adult type" that develops disease, and "Dental type" in which symptoms are limited to the teeth, such as shortened limbs, club knee, fracture, bone deformity, short stature, convulsions, and early deciduous teeth. Symptoms are present. Hypophosphatasia has a high fatality rate, and it is said that the perinatal one-year mortality rate is almost 100%, and the infant-type one-year mortality rate is about 50%.

  Hypophosphatasia, unlike phenylketonuria, another inborn error of metabolism, differs from essential amino acid metabolism abnormalities that can be treated with restricted intake. Their responsible enzyme, TNSALP, is 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 initiation of enzyme replacement therapy results in clinical improvements such as improved patient quality of life and prevention of onset. For this reason, it is desired that these diseases be detected early by a newborn mass screening test.

  In the newborn mass screening test, filter paper that has been soaked in blood and dried (hereinafter referred to as “dry blood filter paper”) is used as a test sample, and tandem mass analysis, enzyme immunoassay (ELISA), enzyme method The presence or absence of dozens of inborn errors of metabolism is detected by high-performance liquid chromatography (HPLC). However, since these existing mass screening methods cannot specifically measure the enzyme activity of TNSALP, no examples have been reported for use in testing for hypophosphatasia.

  Alkaline phosphatase (hereinafter referred to as “ALP”) in the living body is large in the order of small intestinal mucosa, placenta, mammary gland, kidney, bone, lung, liver, and spleen, and is small in skeletal muscle and connective tissue. ALP enzyme activity in serum is derived from ALP produced in these tissues. ALP is broadly classified into non-organ-specific bone type, liver type, kidney type, and organ-specific small intestine type, placenta type, and germ cell type, depending on the gene. TNSALP is an organ non-specific ALP. Serum contains non-organ-specific and organ-specific ALP, and is measured as the total enzyme activity of these six types of ALP isozymes.

  In recent years, bone-type alkaline phosphatase (BAP) has attracted attention as a bone formation marker, and an enzyme immunoassay method (ELISA method) using a BAP-specific antibody to specifically measure BAP in serum, A chemiluminescent enzyme immunoassay (CLEIA method) has been developed and marketed as a measurement kit. However, these measurement kits are expensive and require a dedicated automatic analyzer, which is not suitable for mass screening inspection.

Hypophosphatasia is caused by a decrease in TNSALP enzyme activity due to an abnormality in the TNSALP gene. For this reason, it can be easily discriminated by confirming by measurement that the total ALP enzyme activity in serum is decreased.

The ALP enzyme activity in serum is generally measured by a biochemical automatic analyzer using a measurement method using paranitrophenyl phosphate (hereinafter referred to as “p-NPP”) as a substrate. However, it is easily affected by hemoglobin such as hemoglobin and is not suitable for measurement with a whole blood sample or a hemolyzed sample. There is also an enzyme activity method using a fluorescent substrate as a more sensitive substrate. The method using 4-Methylumbelliferyl phosphate (hereinafter referred to as “4MU-Phos”), a synthetic substrate, is widely used as a substrate for ALP enzyme activity and enzyme immunoassay produced by microorganisms. Since -Phos causes autolysis by a divalent metal ion such as iron (II) ion (Fe ²⁺ ), it cannot measure a sample in which hemoglobin coexists.

Magnesium ions (Mg ²⁺ ) are indispensable for the expression of ALP enzyme activity, and coexistence of Mg ²⁺ is indispensable at the time of ALP measurement. Therefore, when measuring samples 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 a method for monitoring a cell culture state by measuring ALP activity in a culture medium of a cell producing alkaline phosphatase using 4MU-Phos or p-NPP.

  In Patent Document 2, L-phenylalanine (inhibitor) that completely inhibits small intestine alkaline phosphatase is used, and a p-NPP substrate solution containing no inhibitor and a P-NPP substrate solution containing an inhibitor are used. A method for specifically measuring small intestine type alkaline phosphatase activity using the difference in ALP activity obtained is described.

  Patent Document 3 describes a method for detecting hypophosphatasia by detecting a mutation in the TNSALP gene by amplifying a TNSALP gene fragment using several kinds of primers as a screening method for hypophosphatasia.

  Non-patent document 1 describes that in hypophosphatasia, a significant decrease in serum ALP activity is observed due to mutations in the TNSALP gene, so that a diagnosis of measuring ALP activity by a general blood biochemical test is effective. Has been.

  Non-Patent Document 2 describes a method for measuring acid phosphatase and alkaline phosphatase in serum using 4MU-Phos.

  Non-Patent Document 3 describes a method for measuring the activity of ALP bound to the surface of cultured cells using p-NPP.

  Non-Patent Document 4 describes enzyme immunoassay (EIA method) and chemiluminescence immunoassay (CLIA method) as methods for specifically measuring bone alkaline phosphatase (BAP) useful as a bone formation marker in TNSALP. ) Is useful.

  Non-Patent Document 5 describes that human-derived ALP also has different reactivity to inhibitors depending on the type of ALP isozyme, similar to animal-derived ALP.

JP2005-58225 JP2003-180296 JP2011-50283

Orphanet Journal of Rare Disease 2007 2:40 CLINICAL CHEMISTRY 1975; 21 (12): 1791-1794 Cell Biology International 2007; 31: 186-190 Modern Media 2012; 58 (5): 143-148 Hokkaido Dental Journal 2012; 32 (2): 202-209

  It is an object of the present invention to provide a method for measuring the enzyme activity of TNSALP using a dry blood filter paper as a sample without requiring a large-scale measuring device or a dedicated device and easily and inexpensively. By using the method according to the present invention, a mass screening test for hypophosphatasia in newborns becomes possible.

  In the newborn mass screening test, a sample extracted from dry blood filter paper is used as a measurement specimen. Conventional measurement methods cannot measure ALP using dry blood filter paper, and thus screening tests for hypophosphatasia cannot be performed. In addition, in the activity measurement using the synthetic substrate of p-NPP and 4MU-Phos, it is necessary to measure the enzyme reaction within 5 to 30 minutes, and in the mass screening test in which the enzyme reaction is performed using a microplate, The enzyme reaction time was too short, and it was not suitable for measuring a large amount of samples at the same time.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that the composition of the extract does not affect the decrease in enzyme activity when extracting a measurement sample from dry blood filter paper or the pH during enzyme activity measurement. I found. Furthermore, the substrate liquid composition which can measure the said extract sample with high sensitivity, and the composition of the reaction stop liquid which stops an enzyme reaction were discovered.

 It was also found that the influence of fluorescent substances and fluorescent inhibitory substances in the extract of dry blood filter paper is reduced by shifting the measurement wavelength of enzyme activity.

  Furthermore, it has been found that the enzyme activity per unit time of each enzyme decreases as the enzyme reaction time increases. And based on this, by setting the measurement conditions (substrate concentration, sample solution amount, substrate solution amount, reaction stop solution amount, enzyme reaction time) that can sufficiently utilize the enzyme reaction time for mass screening examination, A practical substrate solution composition can be set, and the workability of the measurement operation is improved.

  Based on these findings, a method for detecting TNSALP enzyme activity simply, efficiently and inexpensively from a sample extracted from one dry blood filter paper was found, and the present invention was completed.

  That is, the present invention uses a sample extracted from a sheet of dry blood filter paper to measure TNSALP enzyme activity using an inexpensive substrate solution with a reaction time suitable for mass screening test and simple operation. A method for detecting phosphatase disease and a kit for use in the test method are provided.

Accordingly, the present invention includes the following.
[1] A mass screening test method for hypophosphatasia, which simply measures alkaline phosphatase (ALP) enzyme activity from a sheet of dried blood filter, comprising the following steps:
(1) a step of extracting a blood sample from the dried blood filter paper using an extract,
(2) adding a substrate solution containing a synthetic substrate to the extracted blood sample to cause an enzyme reaction;
(3) A step of measuring fluorescence of the product obtained by the enzyme reaction.
[2] The method according to [1], wherein the extract is a buffer solution not containing phosphate ions and citrate ions.
[3] The method according to [1] or [2], wherein the extract contains a surfactant.
[4] The method according to [3], wherein the surfactant is TritonX-100 and is contained at 0.05% to 0.5%.
[5] The method according to any one of [1] to [4], wherein the extract contains 0.02% to 0.1% sodium azide and 0.05% to 0.5% bovine serum albumin (BSA). .
[6] The method according to any one of [1] to [5], wherein the substrate solution is a buffer solution containing a 4-Methylumbelliferyl (4MU) synthetic substrate.
[7] The method according to [6], wherein the 4MU synthetic substrate in the substrate solution is 4MU-Phos, and is contained at 0.1 mM to 1.0 mM.
[8] The method according to [6] or [7], wherein the pH of the substrate solution is 9 to 11.
[9] The method according to [8], wherein the substrate solution buffer is Tris buffer.
[10] The substrate solution contains 1 mM to 30 mM L-phenylalanine (hereinafter abbreviated as “L-Phe”) as a selective inhibitor that inhibits alkaline phosphatase other than tissue non-specific alkaline phosphatase. [6] The method according to any one of [9].
[11] The method according to any one of [6] to [10], wherein the substrate solution contains 0.5 to 2.0 mM MgCl ₂ as an activity enhancer.
[12] The method according to any one of [6] to [11], wherein the substrate solution contains 1 mM to 30 mM sodium succinate as a substrate autolysis inhibitor.
[13] The method according to any one of [6] to [12], wherein the substrate solution contains 0.01 to 0.1% TritonX-100.
[14] In the step (2), the enzyme reaction is stopped by adding a reaction stop solution containing ethylenediaminetetraacetic acid (EDTA) to the enzyme reaction solution, according to any one of [1] to [13] The method described.
[15] The method according to [14], wherein the reaction stop solution is a glycine buffer.
[16] The method according to [15], wherein the pH of the reaction stop solution is 10 to 11.
[17] The method according to any one of [14] to [16], wherein the enzyme reaction is performed at a temperature of 25 to 45 ° C., and then the reaction stop solution is added.
[18] The method according to any one of [1] to [17], wherein the enzyme reaction is performed in 1 to 4 hours.
[19] The method according to any one of [1] to [18], 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.
[20] A kit for use in the inspection method according to [1], comprising:
(1) dry blood filter paper (2) extract,
(3) a substrate solution containing a synthetic substrate,
(4) Reaction stop solution, and (5) Means for fluorescence measurement of the product obtained by the enzyme reaction.
[21] The kit according to [20], wherein the extract is purified water containing 0.1% BSA, 0.1% TrtionX-100 and 0.05% sodium azide.
[22] The substrate solution containing the synthetic substrate is a 100 mM Tris buffer at pH 9-11 containing 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM sodium succinate, 10 mM L-Phe and 1 mM MgCl ₂ . 20] or the kit according to [21].
[23] The kit according to any one of [20] to [22], wherein the reaction stop solution is a 300 mM glycine-NaOH buffer solution (pH 10.6) containing 10 mM sodium EDTA.

  The present invention makes it possible to measure the enzyme activity of TNSALP, which is a responsible enzyme for hypophosphatasia, from a single dry blood filter paper in a newborn mass screening test using dry blood filter paper as a specimen.

  The present invention provides a substrate composition that can be used for mass screening tests by stabilizing the substrate. In mass screening, a large number of specimens are measured using a 96-well microplate. By using the substrate composition according to the present invention, the measurement time is optimized and the complexity of the test is avoided, so that a large number of specimens are measured at a time. The sample can be measured.

  According to the present invention, it is possible to use a dry blood filter paper that has been used for neonatal metabolic abnormalities as it is for newborns from which it is difficult to collect blood as a specimen. For all newborns, if the possibility of hypophosphatasia can be detected, enzyme replacement therapy, gene therapy, bone marrow transplantation, etc. can be performed at an early stage after birth where clinical symptoms have not yet appeared. There is a high possibility that delays and the like can be stopped.

  Furthermore, the present invention can be applied not only to detection of hypophosphatasia in mass screening tests, but also to grasping disease state, determining treatment policy, confirming treatment effect, follow-up observation, monitoring, evaluation of drug development, etc. .

It is the flowchart which illustrated the enzyme activity measuring method in this invention. The relationship between the substrate solution buffer (Tris buffer, 2-ethylaminoethanol buffer (EAE buffer), iminodiethanol buffer (DEA buffer)) and fluorescence intensity (RFU) is shown. , The relationship between 4MU-Phos concentration (mM) and fluorescence intensity (RFU) in the substrate solution is shown. (In the figure, 4MU-Phos is written as 4MU-P) The relationship between the metal chelating agent (EDTA, sodium citrate, sodium succinate) added to the substrate solution and fluorescence intensity (RFU) is shown. The relationship between the addition concentration (mM) of citric acid and succinic acid to the substrate solution and the fluorescence intensity (RFU) is shown. The relationship between levamisole (levamisole) and L-Phe added to the substrate solution and enzyme activity (pmol / hr / disk) is shown. The relationship between L-Phe concentration (mM) and fluorescence intensity (RFU) in the substrate solution is shown. The relationship between the concentration (μM) of the standard product (4MU) used in the present invention and the fluorescence intensity (RFU) is shown. In the measurement of the present invention, it was used as a calibration curve. The relationship between the enzyme activity (pmol / hr / disk) of TNSALP and the enzyme reaction time (hr) is shown. The distribution diagram of TNSALP enzyme activity in the dried blood filter paper of 2,659 newborns is shown. Arrows indicate TNSALP enzyme activity (pmol / hr / disk) in dry blood filter paper of hypophosphatasia patients.

(Detailed description of the invention)
The present invention is a method for extracting blood containing TNSALP from one sheet of dried blood filter paper and measuring the enzyme activity in the dried blood filter paper using the extracted blood sample.

  Measurement according to the method of the present invention includes (1) a step of extracting a blood sample from a dry blood filter paper using an extract, (2) a step of adding a substrate solution containing a synthetic substrate to the extracted sample, and causing an enzyme reaction to be performed, (3) The process obtained by measuring the fluorescence of the product obtained by the enzyme reaction is completed in three steps.

In the measurement of enzyme activity of blood samples, a composition with a buffer capacity of optimum pH in either or both of the extract solution and the substrate solution so that when the extract sample and the substrate solution are mixed, the pH is suitable for enzyme activity measurement. It is necessary to.

  In the present invention, an extract composition that can effectively extract an enzyme from a dry blood filter paper without interfering with the enzyme activity measurement of TNSALP can be found, and TNSALP activity can be measured from a single dry blood filter paper. It was.

  The extract obtained by the method of the present invention is a buffer solution having a low buffer capacity that does not inhibit the optimum pH of the enzyme reaction when mixed with the substrate solution, and preferably contains phosphate ions or citrate ions that interfere with the ALP enzyme reaction. It is a buffer solution that does not contain.

  The extract according to the method of the present invention does not contain ions interfering with the enzyme reaction, and in order to stabilize the extracted enzyme, a diluted protein such as bovine serum albumin (hereinafter referred to as “BSA”) is allowed to coexist with pH. In addition to providing buffering capacity, the enzyme to be measured is present on the cell membrane on the cell surface, so a surfactant (such as TritonX-100) that acts to crush the cell membrane and release the enzyme into the extract during extraction coexists. It is preferable to make it.

  The extract according to the present invention does not act on the inhibition of enzyme activity to be measured, coexists with an additive having an action of inhibiting nonspecific reaction due to enzyme activity other than the target in the process of extracting the enzyme, and is a dry blood filter paper It is preferable to coexist with a surfactant for promoting extraction from the mixture.

  The surfactant to be added to the extract according to the present invention generally needs to be added at a concentration that does not inhibit the enzyme activity in order to suppress or enhance the enzyme reaction depending on the concentration at which it acts.

  The composition of the extract of one embodiment of the present invention is “purified water containing 0.1% BSA, 0.1% TrtionX-100, 0.05% sodium azide”. BSA in the extract gives the extract a weak pH buffering capacity and acts to protect the ALP enzyme activity. Sodium azide is added for the antiseptic effect of the extract while preventing non-specific reactions due to peroxidases present in the blood. TritonX-100, a surfactant, is added to accelerate the elution of blood components from dry blood filter paper and the elution of enzymes by crushing cell membranes, and shorten the extraction time. The concentration of each additive is preferably set as a concentration that does not inhibit enzyme activity.

  The substrate solution according to the present invention can measure an enzyme using a blood sample extracted with the extract by using a buffer solution having a buffer capacity in a pH range suitable for ALP enzyme activity measurement.

The synthetic substrate used in the substrate solution needs to be a substrate having a structure that becomes a specific substrate of the enzyme to be measured. By using such a synthetic substrate, the reaction specificity with the target enzyme can be improved. However, in the extracted blood sample, innumerable enzymes and ALP isozymes that have similar reactions are present, and therefore coexistence of similar enzyme inhibitors for further increasing the specificity is preferable.

The substrate solution of one embodiment of the present invention has the following substrate solution composition. That is, the substrate solution used for TNSALP enzyme activity measurement (hereinafter referred to as “TNSALP substrate solution”) is “0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM sodium succinate (hereinafter referred to as“ Na succinate ”). Abbreviated)), and a composition of 100 mM Tris buffer having a pH of 9 to 11 containing 10 mM L-Phe and 1.0 mM MgCl ₂ .

  Similar enzyme inhibitor (L-Phe) added to TNSALP substrate solution inhibits ALP isozymes other than TNSALP (small intestine type ALP, placental type ALP, germ cell type ALP), and enhances the specificity of TNSALP enzyme activity measurement Act on. The concentration may be 1 mM or more, and if it is 5 to 30 mM, the enzyme reaction by ALP isozymes can be completely inhibited.

  Since the base buffer solution of the TNSALP substrate solution causes an enzymatic reaction on the alkaline side of pH 9-11, a buffer solution of ethanolamines is generally used, but the ALP concentration in the dried blood filter paper extract sample is a serum sample. Since the activity is measured at a lower concentration compared to the case of using Tris, a trishydroxymethane buffer that can be measured with higher sensitivity is preferable, and a Tris-NaOH buffer having a pH of 10.0 is preferable.

The substrate stabilizer (Na succinate) added to the TNSALP substrate solution does not interfere with the action of Mg ²⁺ necessary for the expression of ALP activity, and is a substrate self-degrading metal such as Fe ²⁺ mixed in from blood samples and other reagents. Add to inhibit some divalent ions. The concentration 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 concentration may be 0.1 mM or more, preferably 0.5 to 3 mM.

  The reaction product produced by the enzyme reaction is 4MU that emits strong fluorescence in the alkaline region. In this reaction, which measures enzyme activity by measuring the fluorescence intensity of this product, highly sensitive detection is possible by stopping the enzyme reaction and adding a reaction stop solution to stabilize the fluorescence. It becomes.

  4MU emits the strongest fluorescence in the alkaline range of pH 9-11. Since the enzyme reaction is carried out in a pH 10 substrate solution, it is not necessary to shift the pH to the alkali side, but a strong metal chelating agent such as ethylenediaminetetraacetic acid (EDTA) is used to stop the ALP enzyme reaction. It is desirable.

  The reaction stop solution contains a metal chelating agent such as EDTA, has a strong buffer capacity in the alkaline region, and is preferably an alkaline buffer solution having a higher concentration than the substrate solution.

  As a reaction stop solution that can be used in one embodiment of the present invention, “300 mM glycine-NaOH buffer solution (pH 10.6) containing 10 mM EDTA” can be mentioned. By using this reaction stop solution, an enzyme reaction can be performed. It can be measured by stopping and enhancing the fluorescence of the reaction product (4MU).

  The reaction product (4MU) absorbs ultraviolet light around 360 nm in an alkaline region exceeding pH 8, and emits fluorescence having a maximum fluorescence intensity around 450 nm.

  The dried blood filter paper extract contains a chromoprotein that interferes with 4MU fluorescence measurement, such as hemoglobin, and a nucleic acid component that absorbs ultraviolet light and inhibits 4MU excitation efficiency. In the present invention, the measurement wavelength is measured by intentionally shifting the maximum excitation wavelength and the maximum fluorescence wavelength of the 4MU in order to reduce the inhibition of the blood component and measure 4MU in the measurement with the reaction solution containing the blood component. .

Due to the shift between the excitation wavelength and the measurement wavelength, the enzyme activity at a low concentration, which could not be measured under the influence of blood components until now, can be measured efficiently, and highly sensitive measurement is possible.

  The wavelength of fluorescence measurement in one embodiment of the present invention is a combination of an excitation wavelength of 370 nm and a detection wavelength of 465 nm.

Enzyme activity is calculated as the amount of 4MU produced per unit time per sheet of dry blood filter paper. The amount of 4MU produced is calculated by comparing the fluorescence intensity of the 4MU standard with the fluorescence intensity of the extracted sample. Moreover, the calculation formula of enzyme activity is calculated by the following formula.
Enzyme activity (pmol / hr / disk) = 4MU amount of extracted sample (μM) x Extracted sample amount (μL) ÷ Enzyme reaction time (hr) x (Extract volume per dry blood filter paper (μL) ÷ Extracted sample Volume (μL))

  In the measurement operation according to an embodiment of the present invention, one dry blood filter paper is put into a well of a microplate, and 200 μL of an extract is added to perform an extraction reaction for 1 hour. 20 μL of the extracted blood sample is transferred to another microplate in a well-to-well manner, 20 μL of the substrate solution is added thereto, and the mixture is reacted at 25 to 45 ° C. for 1 to 24 hours.

  The enzyme reaction time in ALP measurement is generally 5 to 15 minutes. However, in this measurement system, it is possible to measure many samples simultaneously using a microplate. The substrate composition is set so that it can be measured in 1 to 24 hours. The enzyme activity under these measurement conditions is higher than that obtained when the reaction is performed for 24 hours. The enzyme reaction time is preferably 1 to 4 hours. After completion of the enzyme reaction, 200 μL of a reaction stop solution is added to each reaction solution to stop the enzyme reaction. Fluorescence is measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm using a fluorescence microplate auto reader, and the enzyme activity is calculated by comparison 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 comprises the following configuration:
(1) dry blood filter paper (2) extract,
(3) a substrate solution containing a synthetic substrate,
(4) Reaction stop solution, and (5) Means for fluorescence measurement of the product obtained by the enzyme reaction.

  The extract contained in the kit of the present invention is preferably purified water containing 0.1% BSA, 0.1% TrtionX-100 and 0.05% sodium azide.

  The substrate solution containing the synthetic substrate contained in the kit of the present invention preferably has the composition of a TNSALP substrate solution.

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

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

The specimens, reagents, etc. used in the examples are as follows.
[Samples and standard products]
・ Sample: Dry blood filter paper (cut out to φ3mm)
・ 4MU standard product: Product No.M1381 manufactured by SIGMA-ALDRICH

[Synthetic substrate]
・ 4MU-Phos: SIGMA-ALDRICH, Product No.M8883

[Similar enzyme inhibitors]
・ L-Phe: Wako Pure Chemical Industries, reagent special grade

[Substrate stabilizer]
-Sodium succinate (special grade reagent manufactured by Wako Pure Chemical Industries)

・ Extract: Purified water containing 0.1% BSA, 0.1% TrtionX-100 and 0.05% sodium azide ・ Reaction stop solution: 10 mM EDTA, 300 mM glycine-NaOH buffer (pH 10.6)

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

Examination of buffer solution for substrate solution
Substrate buffer was examined using 2-ethylaminoethanol buffer (EAE buffer), iminodiethanol buffer (DEA buffer), and Tris buffer widely used for ALP activity.

(1) Sample, reagent, etc./Sample: Dry blood filter paper (normal human) samples 1, 2, 3
- Substrate solution: 0.2mM 4MU-Phos, 0.05% TritonX-100,10mM L-Phe, 1.0mM MgCl 2, buffer following pH10.0 containing 10mM succinic acid Na (i) ~ (iii) (i) 100 mM Tris buffer (ii) 10% EAE buffer (iii) 10% DEA buffer

(2) Measurement method From a dry blood filter paper, a φ3.0mm dry blood filter paper specimen is cut into each well of a microplate, 200 μL of the extract is added to each well, and extraction is performed for 1 hour while shaking at room temperature. Went. 20 μL of the extracted blood sample was transferred to a Black microplate. 20 μL of the substrate solution was added to the well containing the specimen, and left in a 37 ° C. constant temperature bath for 4 hours to perform the enzyme reaction. After completion of the enzyme reaction, 200 μL of a reaction stop solution was added to each well and mixed gently, and then the fluorescence intensity was measured at an excitation wavelength of 370 nm and a detection wavelength of 465 nm using a fluorescence autoreader.

(3) Results The results are shown in FIG. In the measurement of the present invention in which a sample extracted from dry blood filter paper using 4MU-Phos is measured, a sufficient enzyme reaction can be obtained even with a commonly used ALP measurement buffer (EAE buffer or DEA buffer). The enzyme reaction required for the measurement occurred only with the Tris buffer. For this reason, the TNSALP substrate solution in the present invention was a Tris buffer.

Examination of 4MU-Phos concentration in substrate solution The fluorescence intensity after the enzyme reaction was measured by changing the concentration of 4MU-Phos in the substrate solution in four steps from 0.0625 mM to 0.5 mM.

(1) Sample, reagent, etc./Sample: Dry blood filter paper (normal human) samples 1, 2, 3
- Substrate solution: 0.0625mM~0.5mM 4MU-Phos, 100mM Tris buffer pH10.0 containing 0.05% TritonX-100,10mM L-Phe , 1.0mM MgCl 2, 10mM succinic acid Na

(2) Measurement method Measurement was performed in the same manner as in Example 1, “(2) Measurement method”.

(3) Results The results are shown in FIG. It was found that even when the 4MU-Phos concentration was changed from 0.0625 mM to 0.5 mM, the fluorescence intensity sufficient to measure the dried blood filter paper as a sample was obtained.

Examination of metal chelating agent addition to substrate solution
Coexistence of Mg ²⁺ is essential for the expression of ALP enzyme activity. However, if a large amount of divalent metal ions other than Mg ²⁺ such as Fe ²⁺ derived from hemoglobin is present, autolysis of the synthetic substrate is promoted. For this reason, three types of metal chelating agents (EDTA · 4Na (chelate constant 16.5), sodium citrate (chelate constant 11), and succinate Na (chelate constant 13)) that do not inhibit the effect of Mg ²⁺ to the substrate solution The addition effect of was investigated.

(1) Specimens, reagents, etc. Specimens: Dry blood filter paper (normal human)
・ Substrate solution:
(I) 100 mM Tris buffer solution at pH 10.0 containing 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM L-Phe, 1.0 mM MgCl ₂ (ii) (i) with 4 mM EDTA · 4Na added (Iii) (i) with 4 mM Na citrate (iv) (i) with 4 mM Na succinate

(2) Measurement method Measurement was performed in the same manner as in Example 1, “(2) Measurement method”.

(3) Results The results are shown in FIG. It was found that ALP measurement was inhibited when EDTA · 4Na with the highest chelate constant among the three metal chelating agents was added. On the other hand, it was found that if the citrate constant is lower than that of EDTA · 4Na, sodium citrate or sodium succinate does not inhibit the ALP measurement in this measurement method, and a fluorescence intensity equivalent to that obtained without addition is obtained. It was.

Examination of addition concentration of sodium citrate and sodium succinate to substrate solution TNSALP activity was measured by changing the addition concentration of sodium citrate or sodium succinate in the substrate solution.

(1) Specimens, reagents, etc. Specimens: Dry blood filter paper (normal human)
Substrate solution: 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM L-Phe, 1.0 mM MgCl ₂ , and 100 mM Tris buffer solution at pH 10.0 containing (i) or (ii) below (i) 0 to 8 mM Na citrate
(Ii) 0-8 mM Na succinate

(2) Measurement method Measurement was performed in the same manner as in Example 1, “(2) Measurement method”.

(3) Results The results are shown in FIG. When Na citrate was added, a decrease in activity was observed at a concentration of 4 mM or more, but when Na succinate was added, the activity measurement was not affected. This indicates that sodium succinate is most suitable as a substrate stabilizer.

Examination of levamisole and L-Phe addition effect to substrate solution
The effect of adding levamisole (Levamisole), a known TNSALP inhibitor, and L-Phe, known as an ALP inhibitor, such as the small intestine type, placenta type, and germ cell type, to the substrate solution was examined.

(1) Specimen, Reagent, etc./Sample: Dry blood filter paper (normal newborn dry blood filter paper n = 74)
・ Substrate solution:
(I) 100 mM Tris buffer solution at pH 10.0 containing 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM Na succinate, 1.0 mM MgCl ₂ (ii) (i) with 0.3 mM levamisole added ( iii) (i) plus 10 mM L-Phe

(2) Measuring method
The measurement was performed in the same manner as in Example 1 “(2) Measurement method” except that the enzyme reaction time in a 37 ° C. thermostat was set to 1 hour.

(3) Results The results are shown in FIG. The levamisole concentration required to inhibit TNSALP by 50% is known to be 0.03 mM, and it is considered that TNSALP is sufficiently inhibited when 0.3 mM is used. This revealed that most of the neonatal blood was TNSALP activity inhibited by levamisole. On the other hand, L-Phe is known to have a TNSALP 50% inhibitory concentration of 31 mM, small intestine type 0.8 mM, placental type 1.1 mM, and germ cell type 0.8 mM. The addition of Phe is considered to completely inhibit small intestine type, placental type, and germ cell type ALP. Therefore, it was revealed that TNSALP activity can be measured by adding 10 mM L-Phe to this measurement system.

Examination of L-Phe Concentration of Substrate Solution The fluorescence intensity after the enzyme reaction was measured by changing the L-Phe concentration of the substrate solution.

(1) Specimens, reagents, etc. Specimens: Dry blood filter paper (normal human) samples 1, 2, 3
-Substrate solution: 100 mM Tris buffer solution at pH 10.0 containing ₂ mM 4MU-Phos, 0.05% TritonX-100, 10 mM Na succinate, 0-8 mM L-Phe, 1.0 mM MgCl2.

(2) Measurement method Measurement was performed in the same manner as in Example 1, “(2) Measurement method”.

(3) Results The results are shown in FIG. As described above, the 50% inhibitory concentration of L-Phe, an ALP inhibitor, is 31 mM for TNSALP, 0.8 mM for the small intestine type, 1.1 mM for the placenta type, and 0.8 mM for the germ cell type. From this measurement, it was found that it is desirable to set the concentration within a range that is lower than the 50% inhibitory concentration of TNSALP (31 mM) and higher than twice the 50% inhibitory concentration of ALP other than TNSALP.

Based on the results of Examples 1 to 6, the TNSALP substrate solution in the present invention was prepared as “pH 9 to 11 containing 0.2 mM 4MU-Phos, 0.05% TritonX-100, 10 mM Na succinate, 10 mM L-Phe, 1.0 mM MgCl ₂ . Of 100 mM Tris buffer ”.

Examination of enzyme reaction time Using dry blood filter paper as a sample, TNSALP enzyme activity was detected by changing the enzyme reaction time.
(1) Specimens, Reagents, etc. Specimens: Normal human dry blood filter paper (65 newborn dry blood filter paper)
・ Substrate solution: TNSALP substrate solution (pH 10.0)

(2) Measurement operation
Measurement was performed in the same manner as in Example 1 “(2) Measurement method” except that the enzyme reaction time in a 37 ° C. thermostat was changed stepwise from 1 to 24 hours.

  The measurement result of the fluorescence intensity of the 4MU standard product is shown in FIG. FIG. 9 shows the relationship between the enzyme activity results calculated from the calibration curve and the enzyme reaction time. The enzyme activity of TNSALP decreased with the lapse of the reaction time, showing the highest activity when the enzyme reaction was completed in 1 to 2 hours, and decreased by 10% at 4 hours than when reacted at 1 hour. TNSALP is easily inactivated at high temperatures, and the enzyme is deactivated as the enzyme reaction becomes longer. This is also the reason why the conventional enzyme reaction with serum is set to be measured in 5 to 10 minutes. From these results, it was shown that the method of the present invention can be measured by a reaction for 1 to 4 hours and can be used as a mass screening test method for measuring a large amount of samples using a microplate.

Comparison of patient sample and normal human sample Using dry blood filter paper as a sample, TNSALP enzyme activity was compared between hypophosphatasia patients and normal people.

(1) Samples, reagents, etc.
Normal person (newborn dry blood filter paper) 2,659 cases Low phosphatase patient (dry blood filter paper) case 1 ・ Substrate solution: TNSALP substrate solution (pH 10.0)

(2) Measurement method Measurement was performed in the same manner as in Example 1, “(2) Measurement method”.

(3) Results FIG. 10 shows the distribution of normal human enzyme activity and the enzyme activity of hypophosphatasia patients. The average TNSALP enzyme activity in normal neonates was 914.0 pmol / hr / disk, the standard deviation was 329.0 pmol / hr / disk, and the enzyme activity in hypophosphatasia patients was 103.7 pmol / hr / disk, clearly indicating that normal TNSALP enzyme The activity was lower than the activity. Therefore, the measurement of the present invention showed that a screening test for hypophosphatasia can be performed using dry blood filter paper.

  The measurement method according to the present invention can be used in a newborn mass screening test or the like.

Claims (23)

A mass screening test method for hypophosphatasia, wherein the alkaline phosphatase (ALP) enzyme activity is simply measured from one sheet of dry blood filter paper, the method comprising the following steps:
(1) a step of extracting a blood sample from the dried blood filter paper using an extract,
(2) adding a substrate solution containing a synthetic substrate to the extracted blood sample to cause an enzyme reaction;
(3) A step of measuring fluorescence of the product obtained by the enzyme reaction.   The method according to claim 1, wherein the extract is a buffer solution not containing phosphate ions and citrate ions.   The method according to claim 1 or 2, wherein the extract contains a surfactant.   4. The method of claim 3, wherein the surfactant is Triton X-100 and is included at 0.05% to 0.5%.   The method according to any one of claims 1 to 4, wherein the extract contains 0.02% to 0.1% sodium azide and 0.05% to 0.5% bovine serum albumin (BSA).   The method according to any one of claims 1 to 5, wherein the substrate solution is a buffer solution containing a 4-Methylumbelliferyl synthetic substrate.   The method according to claim 6, wherein the 4-Methyumbelliferyl synthetic substrate in the substrate solution is 4-Methylumbelliferyl phosphate and is contained at 0.1 mM to 1.0 mM.   The method according to claim 6 or 7, wherein the pH of the substrate solution is 9 to 11.   9. The method of claim 8, wherein the substrate solution buffer is Tris buffer.   10. The method according to claim 6, wherein 1 to 30 mM of L-phenylalanine is contained in the substrate solution as a selective inhibitor that inhibits alkaline phosphatase other than tissue non-specific alkaline phosphatase. The method according to any one of claims 6 to 10, wherein the substrate solution contains 0.5 to 2.0 mM MgCl ₂ as an activity enhancer.   The method according to any one of claims 6 to 11, wherein the substrate solution contains 1 mM to 30 mM sodium succinate as a substrate autolysis inhibitor.   The method according to any one of claims 6 to 12, wherein the substrate solution contains 0.01 to 0.1% TritonX-100.   The method according to any one of claims 1 to 13, wherein in the step (2), the enzyme reaction is stopped by adding a reaction stop solution containing ethylenediaminetetraacetic acid (EDTA) to the enzyme reaction solution.   The method according to claim 14, wherein the reaction stop solution is a glycine buffer.   The method according to claim 15, wherein the pH of the reaction stop solution is 10 to 11.   The method according to any one of claims 14 to 16, wherein the reaction stop solution is added after the enzyme reaction is performed at a temperature of 25 to 45 ° C.   The method according to any one of claims 1 to 17, wherein the enzyme reaction is performed in 1 to 4 hours.   The method according to any one of claims 1 to 18, wherein a 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 kit for use in the inspection method according to claim 1, comprising:
(1) dry blood filter paper (2) extract,
(3) a substrate solution containing a synthetic substrate,
(4) Reaction stop solution, and (5) Means for fluorescence measurement of the product obtained by the enzyme reaction.   The kit according to claim 20, wherein the extract is purified water containing 0.1% BSA, 0.1% TrtionX-100 and 0.05% sodium azide. The substrate solution containing the synthetic substrate is a 100 mM Tris buffer having a pH of 9 to 11, containing 0.2 mM 4-methylumbelliferyl-phosphate, 0.05% TritonX-100, 10 mM sodium succinate, 10 mM L-phenylalanine and 1 mM MgCl _2. The kit according to 20 or 21.   The kit according to any one of claims 20 to 22, wherein the reaction stop solution is a 300 mM glycine-NaOH buffer solution (pH 10.6) containing 10 mM sodium EDTA.