Method for quantifying phenylalanine in blood, and a measurement kit used therefor.

Abstract

The present invention provides a method for simply and quickly quantifying phenylalanine (Phe) contained in blood, and a measurement kit used for the method. A quantification method according to the present invention uses a test piece that is made of at least two sheet-like members (first member and second member), and comprises the following steps: (A) a step for dripping blood on a sample holding layer of the first member of the test piece in a state separated from the second member, and allowing to stand for a predetermined period of time; (B) a step for layering the first member on the second member such that the sample holding layer of the first member and an indicator layer of the second member overlap in a non-contact state, dripping a solution containing phenylalanine ammonia-lyase on the sample holding layer, and allowing to stand for a predetermined period of time; (C) a step for measuring the ammonia concentration from the color of the indicator layer of the second member; and (D) a step for calculating the Phe concentration in blood from the ammonia concentration.

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying the concentration of phenylalanine in the blood. The present invention also relates to a measurement kit used in the said method. Furthermore, the present invention relates to a method for easily checking and managing the blood phenylalanine concentration in patients with phenylketonuria using the said method or the said kit, either at the bedside or in a location other than a medical institution. [Background technology]

[0002] Phenylketonuria is a congenital amino acid metabolism disorder in which phenylalanine (hereinafter also referred to as "Phe") cannot be metabolized to tyrosine due to a decrease in the activity of phenylalanine hydroxylase (hereinafter also referred to as "PAH") in the body, resulting in the accumulation of Phe in the body. If a significant increase in Phe concentration persists during infancy, it can lead to severe intellectual disability. By controlling the blood Phe concentration within a certain range, intellectual disability can be prevented and normal intellectual development can be achieved. Therefore, patients with phenylketonuria need to adjust their Phe intake through dietary therapy, using blood Phe concentration as a biomarker. Since blood Phe concentration is easily affected by the amount of Phe contained in food, and Phe tolerance depends on the individual's PAH activity, it is necessary to adjust the amount of Phe intake precisely by repeatedly measuring blood Phe concentration regularly or irregularly. For this purpose, it is necessary that blood Phe concentration can be measured simply and quickly at the patient's bedside or in places other than medical facilities in daily life.

[0003] Currently, the only method available in clinical practice for measuring blood phenylethylamine (Phe) concentration is HPLC. However, this method requires at least one week to obtain results, making it unsuitable for situations requiring immediate evaluation, such as adjusting Phe intake in dietary therapy. On the other hand, mass spectrometry, used in newborn mass screening, is a method that allows for rapid measurement. However, this method requires sample pretreatment and large analytical equipment, making it unsuitable for testing outside of medical institutions.

[0004] Furthermore, conventional methods for diagnosing phenylketonuria include administering Phe labeled with carbon isotopes orally or by injection to a subject and diagnosing phenylketonuria based on changes in the amount of carbon isotopes in the exhaled breath (Patent Document 1), and quantifying L-phenylalanine in a biological sample by using a dehydrogenase, coenzyme, electron mediator, and tetrazolium salt specific to L-phenylalanine in a biological sample as reaction reagents to generate formazan through enzymatic and redox reactions between the biological sample and the reaction reagents, and detecting it optically and / or electrochemically (Patent Document 2). However, the former method has the problem that a mass spectrometer is required to measure the amount of carbon isotopes. On the other hand, the latter method is a simple and rapid method for testing for phenylketonuria, but it is a method that can simultaneously test for three congenital metabolic disorders: galactosemia, maple syrup urine disease, and phenylketonuria, and its principle differs from the measurement method of the present invention described later. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 9-257791 [Patent Document 2] International Publication No. 02 / 018627 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of Biotechnology 258(2017) p.148-157 [Non-Patent Document 2] Guidelines for the Treatment of Diseases Targeted by Neonatal Mass Screening 2019, Diagnosis and Treatment Co., Ltd., edited by the Japanese Society for Inborn Errors of Metabolism, p. 16. [Non-Patent Document 3] Moat, SJ et al. J Inherit Metab Dis 43, 179-188 (2020). [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] As mentioned above, there has been a long-standing need for a simple and rapid method for measuring blood Phe concentration. Therefore, the present invention aims to provide a simple and rapid method for measuring blood Phe concentration. The present invention also aims to provide a measurement kit used in the above method. Furthermore, the present invention aims to provide a method for managing blood Phe concentration in patients with phenylketonuria. [Means for solving the problem]

[0008] The inventors noticed that when Phe is decomposed with phenylalanine ammonia lyase (hereinafter also referred to as "PAL"), an amount of ammonia equal to Phe is produced, and they hypothesized that the Phe concentration in venous blood could be quantitatively measured using this ammonia production amount as an indicator. However, through continuous research, they encountered a problem: when using capillary blood collected from fingertips, etc., as the test sample (test blood), ammonia contained in sweat, etc., was mixed in, making it impossible to accurately measure the Phe concentration in the subject's venous blood. Therefore, in order to solve this problem, they continued their diligent research and confirmed that by first coexisting the collected capillary blood with an alkaline buffer, gasifying and volatilizing the ammonium ions contained therein, and then reacting it with PAL, it was possible to accurately measure the Phe concentration in venous blood even when using capillary blood as the test blood. Furthermore, even when using venous blood as the test blood, removing the inherent ammonium ions by gasification beforehand allows for even more accurate measurement of the subject's blood Phe concentration. This invention was completed through such accumulated research and has the following embodiments.

[0009] (I) Method for determining blood Phe concentration (I-1) A method for quantifying the blood Phe concentration of a subject using a test piece consisting of at least two sheet-like members, The aforementioned test specimen is formed by laminating a first sheet-like member (hereinafter sometimes abbreviated as "first member") having a sample-holding layer containing an alkaline buffer and a second sheet-like member (hereinafter sometimes abbreviated as "second member") having an indicator layer that changes color upon reaction with ammonia gas, in a peeled or peelable state. The aforementioned quantitative method is a method comprising the following steps (A) to (D): Method for quantifying blood Phe concentration: (A) A step of dropping blood (test blood) collected from a subject, either as is or after dilution, and separated from the second component, onto the sample holding layer of the first component of the test piece, and leaving it for a certain period of time. (B) Laminating the first member onto the second member such that its sample holding layer and the indicator layer of the second member overlap in a non-contact state, dropping a PAL-containing liquid onto the sample holding layer and leaving it for a certain period of time. (C) A step of measuring the ammonia concentration in the test blood from the degree of color of the indicator layer of the second component, and (D) A step of calculating the blood Phe concentration of the subject from the ammonia concentration. (I-2) The alkali buffer in the sample holding layer of the first member is an alkali buffer containing boric acid and sodium hydroxide. The method for quantifying blood Phe as described in (I-1), wherein the indicator in the indicator layer of the second member is bromocresol green. (I-3) The content of boric acid and sodium hydroxide in the sample holding layer of the first member is 0.40 to 0.45 mg and 0.15 to 0.20 mg per member, The bromocresol green content in the indicator layer of the second component is 0.3 to 0.5 mg per component. In step (A) above, the amount of test blood dropped onto the sample holding layer is at least 10 μl. The amount of PAL in the PAL-containing solution dropped onto the sample holding layer in step (B) above is at least 2 mU. The blood Phe concentration that can be calculated in step (D) above is 0 to 1400 μM, preferably 0 to 437 μM. (I-2) Method for quantifying blood Phe as described above. (I-4) A method for quantifying phenylalanine in blood as described in (I-2) or (I-3), wherein step (C) is a step of measuring the absorbance of the color of the indicator layer at a wavelength of 635 nm. (I-5) A method for quantifying blood Phe as described in any of (I-1) to (I-4), wherein the blood collected from the subject (test blood) is capillary blood. (I-6) A method for quantifying blood Phe as described in any of (I-1) to (I-5), wherein the subject is a patient with phenylketonuria. (I-7) A method for quantifying blood Phe concentration as described in any of (I-1) to (I-6), wherein the method for quantifying blood Phe is performed using the kit described in (II-1) below. These quantification methods can also be rephrased as an estimated quantification method of Phe in blood or an estimation method of the Phe concentration in blood.

[0010] (II) Blood phenylalanine measurement kit (II-1) A kit containing at least the following (a) and (b), or (a) to (c) for use in the method for quantifying Phe in blood described in any one of (I-1) to (I-6): (a) A first member having a sample holding layer containing an alkaline buffer, and a second member having an indicator layer that discolors upon reaction with ammonia gas, the test piece being formed by laminating the two members in a peeled state or a peelable state. (b) PAL, and (c) A solvent.

[0011] [[ID=1 / 4]] (III) Methods for managing blood Phe concentration (III-1) A method for managing the Phe concentration in the blood of phenylketonuria patients, At a location other than a medical institution, either regularly or irregularly, using capillary blood of a phenylketonuria patient as the test blood, and performing the method for quantifying Phe in blood described in any one of (I-1) to (I-6). <( (III-2) The management method according to (III-1), which is a method of performing the method for quantifying Phe in blood using the kit described in (II-1).

Advantages of the Invention

[0012] According to the method of the present invention, the Phe concentration in the blood of a subject can be measured simply and quickly at the blood collection site. Further, according to the method of the present invention, even when a small amount of blood (capillary blood) from, for example, a fingertip is used as the test blood, the Phe concentration in the venous blood or plasma of the subject can be determined with high accuracy. Therefore, the method of the present invention can be effectively used for screening for early detection of phenylketonuria in newborns, monitoring the Phe concentration in the blood at the bedside of phenylketonuria patients, and monitoring and managing the Phe concentration in the blood, particularly in dietary therapy, at locations other than medical institutions in daily life.

Brief Description of the Drawings

[0013] [Figure 1] An example of specimen 1 is shown in a perspective view. [Figure 2] An example of specimen 1 is shown in cross-sectional view AA. [Figure 3] This shows a cross-sectional view of a sample of specimen 1. [Figure 4] This figure shows the peeling operation of the first sheet-like member 2 and the second sheet-like member 3 of the test specimen 1, and the state after both have been peeled apart. [Figure 5] This figure shows step (A) of the quantitative method of the present invention. [Figure 6] This figure shows step (B) of the quantitative method of the present invention. [Figure 7] This figure shows step (C) of the quantitative method of the present invention. [Figure 8] A schematic diagram illustrating the measurement principle of the present invention is shown. [Figure 9] The cross-sectional view of the AMMONIA TEST KIT II AmiCheck® reaction test paper (AmiCheck) used in the experimental example is shown. [Figure 10] In Reference Experiment Example 1, the results of measuring the ammonia concentration (μM) in capillary blood (test blood) collected from the fingertip are shown using AmiCheck and an ammonia meter. [Figure 11] In Reference Experiment Example 2, the relationship between the time (0-10 minutes) after dropping capillary blood (test blood) collected from a fingertip onto the sample holding layer of the spacer portion of the AmiCheck and the measured ammonia concentration in the test blood is shown (Y axis [AU] is "measured value at each standing time / measured value at 0 minutes of standing time"). [Figure 12] In Experimental Example 1, venous blood (test blood) collected from healthy individuals (3 individuals: A-C) was mixed with Phe at known concentrations (μM) (X axis) (test samples (A)-(C)), and the ammonia concentration (μM) (Y axis) in the test samples was measured using AmiCheck and an ammonia meter. The results are shown below. [Figure 13]In Experimental Example 2, the figure shows a correlation between the ammonia concentration (μM) in the capillary blood of a patient with phenylketonuria, measured using AmiCheck and an ammonia meter (Y axis), and the plasma Phe concentration of the same patient. [Figure 14] In Experimental Example 3, the capillary blood of a patient with phenylketonuria was used as the test blood, and this figure shows that the plasma Phe concentration predicted from the ammonia value measured by the method of the present invention correlates with the actual plasma Phe concentration of the patient. [Figure 15] This figure shows the correlation between the results of Experiment A (plasma Phe concentration in PKU patients) measured in Experiment Example 4(1) and the results of Experiment B1 (capillary blood Phe concentration in PKU patients), plotted on the X-axis and Y-axis, respectively. [Figure 16] This figure shows the correlation between the results of Experiment B2 (Phe concentration in the test sample), measured in Experiment Example 4(2), plotted on the X-axis and the results of Experiment C2 (ammonia measurement value) plotted on the Y-axis. [Figure 17] In Experimental Example 4(3), the results of Experiment A (plasma Phe concentration of PKU patients) are plotted on the X-axis, and the plasma Phe concentration estimated by fitting the ammonia measurement values ​​measured in Experiment C1 using fingertip capillary blood from the PKU patients into the regression equation derived from Experimental Examples 4(1) and (2) is plotted on the Y-axis, showing the correlation between the two. [Modes for carrying out the invention]

[0014] (I) Method for quantifying blood Phe The present invention provides a method for quantifying blood Phe (hereinafter also referred to as "this quantitative method"), characterized by using a test specimen consisting of at least two sheet-like members (hereinafter referred to as "this test specimen") and performing the steps (A) to (D) described below. The following describes the test specimen and steps (A) to (D).

[0015] (1) This test specimen This test specimen consists of a first sheet-like member (first member) equipped with a sample-holding layer containing an alkaline buffer, and a second sheet-like member (second member) equipped with an indicator layer that changes color upon reaction with ammonia gas. The first member is placed (laminated) on top of the second member. The two members may be separated from each other, or they may be temporarily bonded together in a way that allows them to be separated so as not to fall apart.

[0016] Figures 1-3 show the test specimen. 1 An example is shown. Figure 1 is the test specimen. 1 This is a perspective view. Figure 2 shows the test specimen shown in Figure 1. 1 This is a cross-sectional view taken in the AA direction. Figure 3 shows the specimen shown in Figure 1. 1 This is a cross-sectional view taken in the direction of BB. In these figures, the same parts are denoted by the same reference numerals.

[0017] As shown in the figure, this test specimen 1 First member constituting 2 Sheet-like base material 11 (Hereafter simply referred to as "First Substrate") 11 (Also called) There is a hole 12 A layer is formed, and a sample holding layer is placed on top of it to cover the pore. 13 It is arranged. Also, this test specimen 1 The second component that makes up 3 Substrate on the sheet 14 (Hereafter simply referred to as "Second Substrate") 14 On top of the first substrate (also known as "") 11 hole 12 In the corresponding part, the indicator layer 15 It is positioned, with adhesive layers in front of and behind it, respectively. 16a and 16b The first substrate is arranged. 11 Holes formed in 12 As will be described later, the sample holding layer 13 The ammonia gas produced is used in the second substrate 14 The indicator layer is positioned as such. 15 These are vents designed to allow air to reach a certain point. For this reason, they are also called NH3 vents.

[0018] Figure 1 shows the first member. 2 is the second member 3 This test specimen is stacked on top of the above. 1 This shows the first substrate. In this case, as shown in Figures 2 and 3, 11 hole 12 However, the second member 3 Indicator layer 15 Located above, hole 12 The opening is the indicator layer 15 The second member is covered by 3 First member on top 2 Place the indicator layer. 15 Adhesive layers before and after 16a and 16b is the first member 2 and second member 3 This is a part that allows temporary attachment in a detachable state. It is sufficient that it can be temporarily attached, the first member 2 and Second member 3 The adhesive layer does not necessarily need to be bonded; it may be in a detached state. 16a and 16b The adhesive layer may be formed in a way that allows for adhesion, repeating the process two or more times. 15 It may be formed only in front of or only behind.

[0019] 1st base material 11 The size is not particularly limited, but in the case of a strip shape, for example, it is 20-80 mm in length x 3-10 mm in width x 0.1-0.7 mm in thickness, preferably 30-60 mm in length x 4-9 mm in width x 0.1-0.5 mm in thickness, and more preferably 30-40 mm in length x 5-8 mm in width x 0.1-0.4 mm in thickness. Second base material 14 The size is not limited, however, in the case of a strip shape, the first substrate 11It is preferable that the width is the same but the length is different. For example, the length is 20-100 mm x width 3-10 mm x thickness 0.1-0.7 mm, preferably 40-80 mm x width 4-9 mm x thickness 0.1-0.5 mm, and more preferably 40-60 mm x width 5-8 mm x thickness 0.1-0.4 mm. The material of these components is not particularly limited and examples include polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), polycarbonate (PC), etc. Among these, PET and PC are preferred, and PET is particularly preferred.

[0020] 1st base material 11 Holes formed in 12 Although shown as a circular shape in Figure 1, it is not limited to this and can be any shape, such as a rectangle. The size of the hole is also not limited; for example, in the case of a circular shape, the first substrate 11 Depending on the size (especially the width), the diameter can be set in the range of 2 to 8 mm. Preferably, it is in the range of 3 to 5.5 mm, more preferably 3.5 to 4.5 mm.

[0021] 1st base material 11 The sample holding layer is positioned above. 13 This is a porous material obtained by impregnating a porous material with an aqueous solution containing an alkaline buffer and drying it. Any porous material, whether fibrous or non-fibrous, can be used as the porous material, as long as it is inert to at least the test sample (blood (whole blood)) and the alkaline buffer and does not affect the method of the present invention. Examples of fibrous porous materials include filter paper, nonwoven fabric, and woven fabric. Examples of non-fibrous porous materials include porous materials made from 6-nylon, 6,6-nylon, cellulose acetate, cellulose nitrate, polyethylene, and polypropylene. While not limited, a non-fibrous porous material is preferred. Sample holding layer 13 The size of the first member is 11 Holes formed in 12It is sufficient to be large enough to block the opening, and is not limited to that extent. For example, as shown in Figure 1, the first member 2 If it is in the shape of a strip, the sample holding layer 13 The planar shape is rectangular, and the length of one side (horizontal) is the first base material. 11 It is preferable that the width is the same as the width of the material. For example, the width is 3 to 10 mm x height is 3 to 10 mm x thickness is 0.1 to 0.5 mm, preferably the width is 4 to 9 mm x height is 4 to 9 mm x thickness is 0.1 to 0.4 mm, and more preferably the width is 5 to 8 mm x height is 4 to 9 mm x thickness is 0.1 to 0.3 mm.

[0022] Sample holding layer 13 The alkaline buffering agent to be retained is the sample holding layer. 13 Any alkaline buffer that makes the sample to be spot-applied alkaline, gasifying the ammonium ions contained in the sample to produce ammonia gas, is acceptable and is not particularly limited in that respect. For example, examples include alkaline buffers with a pH of 9 to 11, such as a boric acid buffer prepared from boric acid and sodium hydroxide, a sodium borate buffer prepared from sodium borate and sodium hydroxide, and a carbonic acid-bicarbonate buffer prepared from sodium carbonate and sodium bicarbonate. A boric acid buffer is preferred. The alkaline buffer is, for example, dissolved in ultrapure water to form an alkaline buffer in the sample holding layer. 13 By impregnating it and drying it (e.g., natural drying, air drying, heat drying, vacuum drying, etc.), the sample holding layer is formed. 13 It can be kept in a dry state.

[0023] 2nd base material 14 The indicator layer is positioned on top. 15 The indicator layer contains the indicator described later. 15 This includes any component coated with an indicator, and the sample holding layer 13 Similarly, a porous member may be impregnated with an aqueous solution containing an indicator and then dried. These members only need to be inert to the indicator and ammonia gas and not affect the method of the present invention. Indicator layer 15 The size of the first substrate11 Holes formed in 12 It only needs to be large enough to block the opening, and is not limited to that extent. For example, as shown in Figure 1, the second sheet-like member 3 If it is in the shape of a strip, the indicator layer 15 The planar shape is rectangular, and the length of one side (horizontal) is the second base material. 14 It is preferable that the width is the same as the width of the material. For example, the width is 3 to 10 mm × height is 3 to 10 mm × thickness is 0.01 to 0.3 mm, preferably the width is 4 to 9 mm × height is 4 to 9 mm × thickness is 0.01 to 0.2 mm, and more preferably the width is 5 to 8 mm × height is 4 to 9 mm × thickness is 0.01 to 0.1 mm.

[0024] Indicator layer 15 As the indicator to be maintained, a pH indicator that reacts to pH changes caused by ammonia gas and develops or changes color can be used. Although not limited, bromocresol green is a preferred example. Bromocresol green is a pH indicator that appears yellow at around pH 3.8 but changes to blue at around pH 5.4.

[0025] Second member 3 In the indicator layer, 15 Adhesive layer to be placed before and after 16a and 16b As mentioned above, the first member 2 and second member 3 This is the part that keeps the two parts bonded together in a peelable state. The members constituting the adhesive layer, as well as the adhesive components and bonding means, are not particularly limited as long as they do not affect the method of the present invention. Although not limited, examples of bonding means include using adhesives such as acrylic adhesives and elastomer adhesives, using double-sided tape, and a lamination method by heat fusion. 16 The material may be reusable for two or more applications. In this case, adhesive or double-sided tape can be used as the bonding method.

[0026] adhesive layer 16a and 16bThe size of the second member is not particularly limited. For example, 3 If it is in the shape of a strip, the adhesive layer 16a and 16b The planar shape is rectangular, and the length of one side (horizontal) is the second member 14 It is preferable that the width is the same as the width of the adhesive layer. For example, the width is 3 to 10 mm × the height is 2 to 10 mm × the thickness is 0.01 to 0.3 mm, preferably the width is 4 to 9 mm × the height is 2 to 9 mm × the thickness is 0.01 to 0.2 mm, and more preferably the width is 5 to 8 mm × the height is 2 to 8 mm × the thickness is 0.01 to 0.1 mm. 16 It may be possible to apply it two or more times.

[0027] This test specimen 1 is the first substrate 11 hole 12 However, the second member 3 Indicator layer 15 Located above, hole 12 The opening is the indicator layer 15 The second member is covered by 3 First member on top 2 Place and adhesive layer 16a and 16b It can be manufactured by bonding in a peelable state via [a specific method / concept]. However, as will be explained later, this test specimen 1 In implementing this quantitative method, first, the first component 2 and second member 3 Remove the adhesive (temporary adhesive) from the first member. 2 The second member 3 It is used in a separated state (see Figure 4). Note that the first member 2 and second member 3 If the test specimen is used in which the layers have already been removed, the aforementioned peeling procedure is unnecessary.

[0028] (2) Method for quantifying blood Phe using this test specimen The quantitative method using the aforementioned test specimen can be carried out by the following steps (A) to (D). An example of the series of steps is shown in Figures 5 to 7. (A) The blood collected from the subject (test blood), either as is or after dilution, is used in the second component. 3 The test specimen is in a separated state. 1 First member 2 Sample holding layer 13 The process involves dropping it onto something and letting it stand for a certain period of time. (B) The first member 2 the sample holding layer 13 and second member 3 Indicator layer 15 The second member overlaps in a non-contact state. 3 The sample holding layer is laminated on top of the sample holding layer. 13 The process involves dropping a PAL-containing solution onto the surface and letting it stand for a certain period of time. (C) Second component 3 Indicator layer 15 A step of measuring the ammonia concentration in the test blood based on the degree of color, and (D) A step of calculating the blood Phe concentration of the subject from the ammonia concentration.

[0029] (A) Process (see Figure 5) (A) Step (A) is the process of dropping the test blood onto the sample holding layer of the test piece. The test blood is blood collected from the subject to be measured. The subject is a newborn suspected of having phenylketonuria (a newborn requiring testing) or a patient with phenylketonuria. The blood is whole blood and may be capillary blood collected from a capillary or venous blood collected from a vein. Capillary blood is preferred. More preferably, capillary blood collected from the fingertip is preferred because it is easier to collect. The blood may be applied directly to the sample holding layer as a test sample, but if the Phe concentration in the blood is high or the amount of blood collected is small, it may be diluted with a solvent that does not affect the present invention as needed before being applied to the sample holding layer. Although not limited, one example is capillary blood collected from the fingertip. 40 Add any amount of 50 mM Tris-HCl buffer. 43 Diluted by adding (diluted sample) 44) can also be used as the test sample (see Figure 5). In this case, the dilution ratio is not limited, but can be set appropriately so that the ammonia measurement value obtained in step (C) described later falls within the detection range of the blood ammonia analyzer used for the measurement. This test blood 40 or a diluted sample containing it 44 (These will also be collectively referred to as "test samples" without distinction) are prepared in advance by the second component. 3 The first member is in a separated state. 2 Sample holding layer 13 Add the sample dropwise and leave it at room temperature (1-30°C, the same applies below) for 5 minutes or more. This operation can remove ammonia contained in the test sample. Specifically, the test sample is placed in the sample holding layer 13 When dropped into the sample, the alkaline buffer in the sample holding layer dissolves, making the test sample alkaline, and the ammonium ions contained in the test sample gasify. The gasified ammonia gas is removed by volatilization after being left for a certain period of time. The standing time can be any time that allows for the removal and reduction of ammonia gas; for example, at room temperature, 5 minutes or more is sufficient. There is no limit, but preferably 8 minutes or more, more preferably 10 minutes or more. If rapid results are required, it is preferable to keep the standing time within 15 minutes. More preferably, it is about 8 to 10 minutes. Thus the first member 2 Sample holding layer 13 It can hold the test sample 45 (ammonia-free test sample) from which ammonia has been removed or reduced.

[0030] (B) Process (See Figures 6 and 7, upper section) (B) The process involves the first member. 2 Sample holding layer 13 Test samples held in place 45 The sample holding layer 13 This is the enzymatic treatment process described above. The enzyme used in this invention is phenylalanine ammonia lyase (PAL). As shown in Figure 8, PAL is an enzyme that reacts with Phe to produce equimolar amounts of ammonia and cinnamic acid. In other words, if Phe is present in the test sample (i.e., test blood), it will react with PAL to produce equimolar amounts of ammonia. PAL can be prepared using E. coli as shown in the examples described later, but it can also be commercially available for convenience. In particular, recombinant strains prepared using E. coli are easy to handle because they have good stability, and their specific activity does not decrease significantly even after freeze-drying and subsequent storage at room temperature, as shown in the examples section below. PAL can be used or stored in a dissolved state in a solvent. The solvent is not limited as long as it does not impair the activity of PAL, but a 50 mM Tris-HCl aqueous solution is an example. In addition, as long as it does not impair the activity of PAL, an osmotic pressure adjusting agent such as NaCl, a preservative such as sodium azide, and / or a stabilizer such as trehalose may be added to the solvent.

[0031] (B) The enzyme treatment in step (B) is performed on the first member obtained in step (A) above. 2 The sample holding layer 13 However, the second member 3 Indicator layer 15 The second member overlaps in a non-contact state. 3 This is done after lamination on top of the first substrate. 11 hole 12 The opening is in the second member 3 Indicator layer 15 It is covered and sealed. In other words, the enzyme treatment in step (B) is performed on the first member. 2 to the second member 3 The test sample is stacked on top of the other samples. 45 Sample holding layer containing 13 Next, a PAL-containing solution obtained by diluting PAL with a solvent. 47 The substance is added dropwise and left to react for a certain period of time under room temperature conditions. 1While there are no restrictions on the humidity of the environment in which the specimens are left, it is preferable to set and adjust the relative humidity to be within the range of 60-80% RH. Temperature and humidity during the leaving period may be controlled using a constant temperature and humidity chamber, or by placing the specimens in a container or bag containing a desiccant such as silica gel.

[0032] During this standing time, the test sample 45 The Phe derived from the test blood contained in the sample is converted to ammonia by the action of PAL, and these ammonium ions form in the sample holding layer. 13 The alkali buffer inside causes gasification, producing ammonia gas. This ammonia gas is then used in the first substrate. 11 hole 12 (NH3 vent) through the second member 3 Indicator layer 15 It comes into contact with the indicator layer. 15 The indicator reacts and changes color (color-developing portion 49 of the indicator layer). For example, if the indicator is bromocresol green, the color changes from the initial yellow to blue (see upper part of Figure 7).

[0033] During the standing time, the entire amount of Phe contained in the test sample is converted to ammonia by PAL, resulting in the indicator layer as described above. 15 The time required for discoloration is sufficient. Under the above temperature and humidity conditions, 5 minutes or more is sufficient. There is no limit, but preferably 8 minutes or more, more preferably 10 minutes or more. If rapid results are required, the standing time should preferably be kept to within 15 minutes. More preferably around 8 to 10 minutes.

[0034] (C) Process (see Figure 7) (C) Step (C) is the second member obtained in step (B) above 3 Indicator layer 15 This is a step in calculating the ammonia concentration derived from Phe in the test sample from the color (symbol 49). This method involves a blood ammonia measuring device. 5This can be easily implemented using the following device. This device is commercially available, for example, Arkray Corporation's blood ammonia analyzer, PocketChem. TM BA PA-4140 can be mentioned. The measuring device is a compact device (measurement principle: one-wavelength reflectance measurement method, measurement wavelength: 635 nm) developed to measure the ammonia concentration in blood samples using test strips (AMMONIA TEST KIT II AmiCheck®) sold by the company. The cross-sectional structure of the test strip (referred to as "AmiCheck") is shown in Figure 9 (adapted from the AmiCheck accompanying documents). According to the AmiCheck accompanying documents, 20 μL of venous blood is dropped onto the sample holding layer of AmiCheck, and after reacting for 180 seconds, the spacer is peeled off from the base film, and the color-developing portion of the indicator layer of the base film is set in the measurement section of the blood ammonia measuring device. Measurement starts automatically, and after 20 seconds, the ammonia concentration (N-μg / dl) (as nitrogen amount) is displayed on the liquid crystal display. The ammonia measurement concentration range of this device is 6 to 240 μM, which translates to 10 to 400 N-μg / dl in terms of nitrogen amount.

[0035] In step (C) of the present invention, the test specimen treated with enzymes in step (B) is used. 1 Regarding the first member, 2 and second member 3 The indicator layer of the second member 3 is separated. 15 The color-developing portion (49) is set in the measurement section of the blood ammonia measuring device. This automatically displays the ammonia concentration (N-μg / dl) (as nitrogen content) on the liquid crystal display. Note that this ammonia concentration is obtained in the enzyme treatment step (B) of the first member 2 Sample holding layer 13 This reflects the amount of ammonia (Phe-derived ammonia) produced from Phe in the test sample due to the action of PAL within the sample holding layer. 13 It correlates with the amount of Phe contained in the test sample dropped onto the sample. If the test sample is blood diluted with a solvent, the ammonia concentration corresponding to the amount of Phe in the test blood can be determined by converting it to the dilution ratio.

[0036] (D) Process Step (D) is a step in which the subject's blood Phe concentration is calculated from the Phe-derived ammonia concentration contained in the test blood obtained in step (C). Here, blood Phe concentration includes both the Phe concentration in the subject's venous blood (whole blood) and the Phe concentration in its plasma. It is known that the Phe concentration in whole blood is about 20% less than the Phe concentration in plasma. Therefore, if one Phe concentration can be calculated, the other Phe concentration can be calculated automatically.

[0037] The concentration of Phe in the subject's blood can be calculated from the Phe-derived ammonia concentration in the test sample using a calibration curve created in advance through experiments or a function (regression equation) obtained from that calibration curve.

[0038] Calibration curves can be created depending on the origin of the test blood (venous blood, capillary blood), for example, as follows: [When using venous blood as test blood] (a) Multiple test samples are prepared by gradually adding a known amount of Phe to venous blood (whole blood) collected from a healthy individual. The steps (A) to (C) described above are performed on each sample, and the Phe-derived ammonia concentration in the test sample is determined from the degree of color development of the indicator layer of the test piece. (b) Create a calibration curve by plotting the Phe concentration added to venous blood on the x-axis (or y-axis) and the Phe-derived ammonia concentration obtained in (a) on the y-axis (or y-axis).

[0039] [When using capillary blood as test blood] (a) Capillary blood (whole blood) collected from the fingertips of multiple subjects is used as the test sample, and the steps (A) to (C) described above are performed to determine the Phe-derived ammonia concentration in the test sample from the degree of color development of the indicator layer of the test piece. (i) Separately, the Phe concentration in the subject's venous blood (whole blood) is measured by HPLC and the blood Phe concentration is obtained. Create a calibration curve by plotting the blood Phe concentration obtained in (c) and (b) on the x-axis (or y-axis) and the Phe-derived ammonia concentration obtained in (a) on the y-axis (or y-axis). The subjects are preferably individuals whose venous blood Phe concentration is in the range of 0 to 1400 μM, more preferably 0 to 437 μM. They may be patients with phenylketonuria, or healthy individuals who have orally ingested Phe. A mixture of both types of subjects may also be present.

[0040] The regression equation can be calculated from these calibration curves. As shown in Experimental Examples 1-4, the Phe-derived ammonia concentration in the test samples obtained in steps (A)-(C) above correlates very well with the subject's blood Phe concentration (or plasma Phe concentration), and the subject's blood Phe concentration estimated from the Phe-derived ammonia concentration in the test samples obtained in steps (A)-(C) (= blood Phe concentration obtained in step (D)) is in close agreement with the actual blood Phe concentration of phenylketonuria patients. Therefore, by using the calibration curve obtained by the above method or the function (regression equation) obtained therefrom, the actual blood Phe concentration of the subject can be estimated with high accuracy from the Phe-derived ammonia concentration in the test sample obtained by performing steps (A) to (C).

[0041] (II) Blood Phe measurement kit The Phe measurement kit of the present invention contains at least (a) and (b), or (a) to (c) below. By using this kit, the quantitative method described above can be easily carried out. (a) A test specimen comprising a first member having a sample-holding layer containing an alkaline buffer and a second member having an indicator layer that changes color upon reaction with ammonia gas, laminated in a peeled or peelable state, (b) PAL, and (c) Solvent.

[0042] (a) The test specimen is this test specimen 1As stated above, the above description can be incorporated herein. For convenience, test strips manufactured by Arkray, Inc. (AMMONIA TEST KIT II AmiCheck®) can be used. (b) From the viewpoint of storage stability, PAL is preferably a lyophilized product. Although not limited, for example, recombinant PAL prepared from E. coli has been confirmed to have sufficient enzyme activity for use in the present invention for at least 5 weeks even when stored at room temperature in a dry state after lyophilization (see the Examples section below). (c) The solvent may be used to dilute the blood used as test blood and / or to dissolve the lyophilized PAL. Although not limited, for example, a 50 mM Tris-HCl aqueous solution (pH 8.5-9.5) or an equivalent buffered aqueous solution may be used.

[0043] In addition, the kit of the present invention may include blood collection capillaries, pipettes, Eppendorf tubes, and instructions for use.

[0044] (III) Methods for managing blood Phe concentration By using the quantitative method described above, the blood Phe concentration in patients with phenylketonuria can be easily evaluated using venous blood or capillary blood as the test blood. Preferably, the test blood is capillary blood collected from the fingertip. As a result, the blood Phe concentration in patients with phenylketonuria can be monitored regularly (for example, daily or at regular intervals) or irregularly at locations other than medical institutions. Therefore, according to the present invention, a method for managing the blood Phe concentration in patients with phenylketonuria can be provided by using the quantitative method described above. This method can be performed using the measurement kit described above. [Examples]

[0045] The present invention will be explained below using experimental examples to aid in understanding its structure and effects. However, the present invention is not limited in any way by these experimental examples. Unless otherwise specified, the following experiments were conducted at room temperature (20±5℃) and under atmospheric pressure conditions. Unless otherwise specified, "%" below means "mass percent" and "parts" means "parts by mass". Furthermore, the following experiments were conducted after obtaining prior approval from the Ethics Committee of the Graduate School of Medicine, Tohoku University (Japan) (Approval number 2020-1-362).

[0046] The test specimens, ammonia meter, and PAL-containing solution used in the following experimental examples are as follows. (1) Test specimen AMMONIA TEST KIT II AmiCheck® Reaction Test Strip (In Vitro Diagnostic Medical Device): Manufactured by Arkray, Inc. In the following experimental example, this is referred to as "Amicheck".

[0047] Figure 9 shows a cross-sectional view of the AmiCheck. Here, the "spacer" corresponds to the "first substrate" of this test specimen. As shown in Figure 9, holes are formed in the spacer, and a sample holding layer is laminated on its upper surface so as to cover these holes. The sample holding layer contains an alkaline buffer (0.426 mg boric acid / sheet, 0.187 mg sodium hydroxide / sheet). Also in Figure 9, the "base film" corresponds to the "second substrate" of this test specimen. An indicator layer is laminated on the base film so as to be laminated in a non-contact state with the sample holding layer through the holes in the spacer. The indicator layer is coated with bromocresol green (0.04 mg / sheet) as an indicator.

[0048] [Principle of measuring whole blood ammonia using Amicheck] When a test sample (blood) is applied to the sample-holding layer on the AmiCheck spacer, the alkaline buffer in the sample-holding layer dissolves, making the test sample alkaline. This causes the ammonium ions in the test sample to gasify into ammonia gas, which passes through the pores of the spacer and migrates to the indicator layer. The ammonia gas that migrates to the indicator layer reacts with the indicator in the indicator layer, causing the color of the indicator layer to change. When the spacer laminated on the base film is peeled off and the indicator layer portion of the base film is placed on the measurement unit of the ammonia meter described below, the degree of color change (color intensity) of the indicator layer is automatically detected and the ammonia concentration (N-μg / dL) (as nitrogen content) is displayed. In this way, the ammonia concentration in a test sample (blood) can be measured using AmiCheck and the ammonia meter.

[0049] (2) Ammonia measuring instrument Blood ammonia measuring device PocketChem TM BA PA-4140: Manufactured by Arkray, Inc. In the following experimental example, this will be referred to as an "ammonia measuring device."

[0050] (3) PAL-containing liquid (a) Fabrication of PAL The cDNA of PAL contained in plasmid (#78286, Addgene, Cambridge, USA) was inserted into a pET21d(+) vector (69743, Novagen Merck Millipore, Germany) using the In-Fusion HD Cloning Kit (TaKaRa, Japan), and a His6 tag was added to the C-terminus. The prepared pET21d(+) was transfected into E. coli HMS174(DE3) strain (69453, Merck Millipore, Germany) and OD was performed. 600After culturing until the pH reached 0.6, 1 mM isopropylthio-α-D-galactopyranoside (9030, TaKaRa, Japan) was added, and incubation was continued at 25°C for another 15 hours. The cultured E. coli was harvested, lysed using BugBuster protein extraction reagent (70584, Novagen Merck Millipore, Germany), and micrococcal nuclease (2900A, TaKaRa, Japan) was added. PAL was collected using HisSpinTrap (28401353, GE Healthcare, UK) and dialyzed three times (1 hour, 2 hours, 1 day) with 50 mM Tris-HCl solution (pH 8.8) using Mini Dialysis Kit with a 1 kDa cut-off (28955964, GE Healthcare, UK). After concentration using Amicon Ultra (UFC500324, Merck Millipore, Germany), the solution was pierced. TM Protein concentrations were measured using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, USA). The proteins were then subjected to electrophoresis on an SDS-PAGE gel and stained with TaKaRa CBB Protein Safe Stain (TaKaRa, Japan), and a single band corresponding to the predicted molecular weight was identified.

[0051] (b) Preparation of PAL-containing solution Using the recombinant PAL prepared in (a) above using E. coli, the following three types of PAL-containing solutions were prepared.

[0052] (i) PAL-containing liquid (No.1) Prepare a PAL-containing solution using ultrapure water to the following concentrations. Store at 4°C until use. PAL 1-3 mg / mL (as protein content) Tris-HCl 50 mM NaCl 154 mM Sodium azide 3.3 mM

[0053] (ii) PAL-containing liquid (No.2) Prepare a PAL-containing solution (for stock) using ultrapure water to the following concentrations, dispense 20 μL into Eppendorf bottles, and store at -20°C until use. Before use, allow the solution to return to room temperature, and dilute to 100 μL with 50 mM Tris-HCl to obtain the PAL-containing solution. PAL 5-15 mg / mL (as protein content) Tris-HCl 50 mM NaCl 770 mM Sodium azide 16.7 mM. In the reference experiment examples and experimental examples described later, the PAL-containing solution prepared by the above method was used (the PAL content in the diluted PAL-containing solution was 1-3 mg / mL (as protein content)).

[0054] (iii) PAL-containing liquid (No.3) Prepare a PAL-containing solution (for lyophilization) to the following concentrations, dispense 100 μL into each Eppendorf bottle, and pre-freeze by freezing at -80°C for 24 hours. Lipo-dry the solution using a freeze-dryer, and store the resulting lyophilized PAL at room temperature until use. Before use, add 100 μL of ultrapure water to dissolve the lyophilized PAL and prepare a PAL-containing solution. Tris-HCl 50 mM PAL 1-3 mg / mL (as protein content) NaCl 154 mM Sodium azide 3.3 mM Trehalose 10 mg / mL.

[0055] (c) Enzyme activity (specific activity) of PAL 96 μL of a 30 mM Phe aqueous solution was placed in a 96-well plate and allowed to stand at 30°C for 6 minutes. Then, 4 μL of the PAL-containing solution prepared above (No. 3, before freezing) was mixed in, and the absorbance at 275 nm was measured at 30°C every 5 seconds for 2 minutes. The specific activity (U / mg) was calculated using the following formula (Non-patent Literature 1: Journal of Biotechnology 258 (2017) 148-157). As a result, the specific activity of the PAL (recombinant E. coli) prepared in (a) above was 0.332 ± 0.014 U / mg.

[0056]

number

[0057] (d) Stability of PAL As shown in Table 1 below, the above-mentioned PAL retains sufficient enzyme activity for use in the present invention even after being freeze-dried and stored in that state (at room temperature) for at least 5 weeks.

[0058] Table 1 shows the results of measuring the specific activity (U / mg) of PAL after storing the freeze-dried PAL prepared in (iii) above at room temperature for 0 days, 1 week, 3 weeks, and 5 weeks, respectively, by adding 100 μL of ultrapure water to dissolve it and obtaining a PAL-containing solution, using the method described in (c) above.

[0059] [Table 1]

[0060] As shown in Table 1, the specific activity of PAL remained above 0.2 mU even after freeze-drying and storage at room temperature for 5 weeks. This confirms that recombinant E. coli PAL is relatively stable in a dry state and can be used in this quantitative method if stored at room temperature for at least 5 weeks.

[0061] Reference Experiment Example 1 Capillary blood samples were collected from the fingertips of six healthy volunteers and used as test blood. The ammonia concentration in the test blood was measured using Amicheck and an ammonia meter. The measurement of blood ammonia concentration was performed according to the method described in the Amicheck package insert. The results are shown in Figure 10. While normal blood ammonia levels in healthy individuals are reported to be 60 μM or less, the average ammonia level obtained was a high 68.20 ± 28.70 μM. Furthermore, variability in the measured values ​​was observed among the test blood samples. One possible reason for this is that when blood is collected from the capillaries in the fingertips, ammonia contained in sweat and dirt on the fingertips may be mixed in, making it impossible to accurately measure the amount of ammonia in the capillary blood. This is consistent with the "Operating Precautions" stated in the AmiCheck package insert, which says, "Do not use fingertip blood, as contamination with sweat or tissue fluid may result in high readings."

[0062] Reference Experiment Example 2 The present invention is a method for indirectly determining a subject's blood Phe concentration by measuring the amount of Phe-derived ammonia produced by reacting PAL with Phe in a test sample. Therefore, if the ammonia concentration in the test sample fluctuates before measurement, it will affect the Phe value to be determined, reducing its reliability.

[0063] Therefore, as a way to solve this problem, we used capillary blood collected from the fingertips of seven healthy individuals (volunteers) as test blood and performed the following procedure. (1) Separate the spacer portion (first member) and the base film portion (second member) of the temporarily bonded mesh check. (2) Drop 10 μL of test blood (whole blood) onto the sample holding layer of the spacer section. (3) Leave the sample in its original state for 0 minutes, 1 minute, 2 minutes, 5 minutes, and 10 minutes under relative humidity of 40-80%RH. Then, place the spacer back on top of the base film and leave it for 180 seconds as described in the AmiCheck package insert to allow the ammonia gas generated in the sample holding layer to react with the indicator layer. (4) After each standing time, the indicator layer of the colored base film is brought to the measuring section of the ammonia meter, and the ammonia measurement value displayed on the ammonia meter is read.

[0064] Figure 11 shows a graph plotted with the time the area was left standing in step (3) above on the X axis, and the ratio of the ammonia measurement value at 0 minutes of standing time to the ammonia measurement value at each standing time (1 minute, 2 minutes, 5 minutes, 10 minutes) (measurement value at each time / measurement value at 0 minutes: AU) on the Y axis. As shown in Figure 11, it was confirmed that 61.2% of the ammonia contained in the test blood was removed by leaving it for 5 minutes after dropping the test blood onto the sample-holding layer of the spacer. Furthermore, it was confirmed that up to 86.7% of the ammonia contained in the test blood was removed by leaving it for 10 minutes. From this, it was confirmed that even if the test blood contains ammonia at the maximum concentration measured in Reference Experiment Example 1, by dropping the test blood onto the sample-holding layer of the spacer and leaving the spacer separated from the base film for a certain period of time, such as 5 to 10 minutes, 61.2 to 86.7% of the total ammonia amount can be removed, and the ammonia content can be reduced to about 14.5 to 26.4 μM. Even if this amount of ammonia remains in the test blood, it has almost no effect on the measurement of the subject's blood Phe concentration.

[0065] Experimental Example 1 Blood samples (venous blood) were collected from the veins of three healthy volunteers (A-C). A Phe solution of known concentration was added to this test blood at a ratio of 5% v / v. The Phe solution was prepared by gradually dissolving Phe in a 50 mM Tris-HCl aqueous solution to achieve concentrations ranging from 0 to 500 μM in the test blood. The test sample, prepared by adding a known amount of Phe to the test blood, was mixed with twice the volume of PAL-containing solution. 20 μL of this solution was dropped onto the sample holding layer of the AmiCheck and left at room temperature for 10 minutes. After that, the spacer was peeled off the base film, and the indicator layer on the colored base film was placed in the measuring section of an ammonia meter. The ammonia measurement value displayed on the ammonia meter was read.

[0066] Figures 12(A)-(C) show the results of plotting the Phe concentration (μM) added to the test blood (3 samples) on the X axis and the ammonia measurement (μM) on the Y axis. As shown in Figure 12, the R of the regression line obtained from the results of (A)-(C) is shown below. 2 These were 0.99, 0.97, and 0.99, respectively. These results confirm that the amount of Phe in a test sample can be indirectly quantified by measuring the amount of ammonia produced by the reaction of venous blood containing Phe with PAL using AmiCheck.

[0067] Experimental Example 2 10 μL of capillary blood (test blood) collected from the fingertips of phenylketonuria (PKU) patients (N=3) was dropped onto the sample holding layer on the spacer of an AmiCheck, which consisted of a spacer (first component) and a base film (second component), and left at room temperature for 10 minutes. After standing, the spacer was placed back in its original position on the base film, and 10 μL of PAL-containing solution was dropped onto the sample holding layer on the spacer and left at room temperature again for 10 minutes. After standing, the indicator layer on the base film, which had developed color, was subjected to the measurement section of an ammonia meter, and the ammonia measurement value displayed on the ammonia meter was read.

[0068] In parallel, plasma samples of blood (venous blood) collected from the veins of the PKU patient were used as test samples, and the concentration of Phe in the plasma was measured (HPLC method) by an external clinical testing company (BML Co., Ltd.). [HPLC method] Measurement reagent: Amino acid kit (Ninhydrin reagent kit for Wako amino acid automated analyzer: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Amino acid analyzer: L-8900 (Hitachi High-Technologies Corporation)

[0069] Figure 13 shows the results of plotting plasma Phe concentration (μM) on the X axis and ammonia measurement values ​​(μM) from an ammonia meter on the Y axis. As shown in Figure 13, the regression equation is Y = 0.1151X + 78.78, and R 2 The value was 0.99. This indicates that the ammonia concentration in the test blood measured by the method of the present invention, using capillary blood from PKU patients as the test blood, correlates very well with the Phe concentration in the plasma of the same PKU patients. This confirms the possibility that the Phe concentration in the blood or plasma of PKU patients can be indirectly calculated by measuring the ammonia concentration in the test blood using the method of the present invention.

[0070] Experimental Example 3 To verify the results of Experimental Example 2, 10 μL of capillary blood was collected from the fingertip of a different PKU patient (N=3) than in Experimental Example 2. The ammonia concentration in the test blood was measured using AmiCheck and an ammonia meter, in the same manner as in Experimental Example 2. In parallel with Experimental Example 2, blood (venous blood) collected from the veins of the same PKU patient was used as the test sample, and the measurement of Phe concentration in the plasma was outsourced to an external company.

[0071] Figure 14 shows the results of plotting the ammonia measurement values ​​(μM) obtained from capillary blood as the test blood, converted to plasma Phe concentration (predicted plasma Phe value) (μM) using the regression equation obtained in Experimental Example 2, with the Y-axis representing the value and the X-axis representing the actual measured plasma Phe concentration (μM). As shown in Figure 14, the R obtained from the regression equation is shown. 2 The value was 0.8794, indicating a good correlation. This result confirms that the blood Phe concentration of a subject can be indirectly calculated by measuring the ammonia concentration in the test blood using the method of the present invention.

[0072] Furthermore, applying the measurable range of the ammonia meter (6-240 μM, 10-400 μg / dL) to the regression equation obtained in Figure 13 of Experimental Example 2, the detectable range of blood Phe concentration is estimated to be 0-1400 μM.

[0073] Experimental Example 4 Experiments A to C, as described in Table 2, were performed, and the accuracy of this quantitative method (Experiment C) (correlation between capillary ammonia concentration and plasma Phe concentration) was evaluated by comparing Experiment A with Experiment B1 and Experiment B2 with Experiment C.

[0074] [Table 2]

[0075] (1) Experiment A and Experiment B1 Venous blood and capillary blood (collected from the fingertip) were collected from PKU patients (N=14), and these were used as test blood samples. The Phe concentration in plasma and capillary blood was measured using the methods shown in Experimental Examples A and B1 in Table 2, respectively.

[0076] The obtained results were plotted with plasma Phe concentration on the X-axis and capillary blood Phe concentration on the Y-axis (Figure 15). The regression equation obtained from this figure is Y = 0.8731X - 12.75, and R 2 The value was 0.99. From this result, it can be seen that the plasma Phe concentration and capillary blood Phe concentration in PKU patients are very well correlated, and that the plasma Phe concentration can be estimated from the capillary blood Phe concentration by using the regression equation described above. It should be noted that the correlation between the plasma Phe concentration and capillary blood Phe concentration in PKU patients has been reported previously (Non-Patent Literature 3), and the above result was consistent with previously reported findings.

[0077] (2) Experiments B2 and C2 Using ultrapure water as the solvent, Phe aqueous solutions were prepared with Phe concentrations of 0 mM, 1.21 mM, 2.42 mM, and 3.63 mM, respectively. Capillary blood collected from the fingertips of healthy individuals was mixed with each of the aforementioned Phe aqueous solutions at a ratio of 5% v / v, and this was used as the test sample (N=105). For the test sample, the Phe concentration was measured by the method of Experimental Example B2 in Table 2, and the NH3 concentration was measured by the method of Experimental Example C2.

[0078] The obtained results were plotted with the Phe concentration on the X-axis and the NH3 concentration (ammonia measurement value) on the Y-axis (Figure 16). The regression equation obtained from this figure is Y = 0.5007X + 50.485, and R 2 was 0.81. From these results, using capillary blood containing Phe as the test sample, the amount of ammonia (NH3 derived from Phe) generated in the reaction with PAL was measured using an amicheck and an ammonia measuring instrument, and the regression equation was applied to the obtained ammonia measurement value, confirming that the Phe concentration in the test sample can be indirectly quantified (estimated).

[0079] (3) Discussion of the results of Experiments A to C Combining the two regression equations Y = 0.8731X - 12.75 and Y = 0.5007X + 50.48 obtained in (1) and (2) above, the following relational expression holds:

Equation

[0080] Substituting the ammonia measurement value obtained by conducting Experiment C1 using capillary blood (N = 11) from PKU patients into X in this relational expression, the plasma Phe concentration (Y) of the venous blood of the patient was calculated. This value (estimated plasma Phe value) was plotted on the Y-axis, and the plasma Phe concentration of the venous blood of the PKU patients obtained in Experiment A was plotted on the X-axis (Figure 17). As shown in Figure 17, R of the regression equation 2 was 0.97. The measurement error of the estimated plasma Phe value with respect to the actual plasma Phe value was on average 9.55% (95% confidence interval 4.7 - 14.4). From this, it was considered that the quantification (estimated quantification) of the plasma Phe value by this quantification method has an accuracy that is sufficient for use in clinical settings or at home.

[0081] Furthermore, applying the measurable range of the ammonia meter (6-235 μM, 10-400 μg / dL) to the regression equation obtained above, the detectable range of blood Phe concentration is estimated to be 0-437 μM. [Industrial applicability]

[0082] In patients with PKU, it is recommended to control plasma Phe concentration within the range of 120-360 μM to prevent complications (Neonatal Mass Screening Target Diseases and Other Clinical Practice Guidelines 2019, Diagnosis and Treatment Co., Ltd., page 16: Non-Patent Literature 2). Therefore, plasma Phe concentrations of 0-1400 μM, preferably 0-437 μM, that can be detected by the method of the present invention are clinically sufficient. In other words, the method of the present invention is a clinically practical method that can be measured simply and quickly, and can therefore be effectively used for screening for the early detection of phenylketonuria in newborns, monitoring of blood Phe concentration at the bedside in patients with phenylketonuria, and monitoring and management of blood Phe concentration in daily dietary therapy. [Explanation of symbols]

[0083] 1: Test specimen of the present invention 2: First sheet-like member of the test specimen of the present invention 3: Second sheet-like member of the test specimen of the present invention 11: Base material of the first sheet-like member (first sheet base material) 12: Holes in the first sheet-like member (NH3 ventilation holes) 13: Sample holding layer of the first sheet-like member 14: Base material for the second sheet-like member (second sheet base material) 15: Indicator layer of the second sheet-like member 16a: Adhesive layer of the second sheet-like member (front part of the indicator layer) 16b: Adhesive layer of the second sheet-like member (rear part of the indicator layer) 40: Test blood 41: Capillary tube for blood collection 42: Eppendorf 43: Solvent 44: Test sample containing test blood and solvent 45: Deammonia-removed test sample after being dropped onto the sample holding layer and left standing. 46: Phenylalanine ammonia lyase (PAL) 47: PAL-containing solution obtained by dissolving PAL in a solvent 48: Reaction spot between test sample containing test blood and PAL-containing solution 49: Indicator layer that has changed color in response to ammonia gas. 5: Ammonia measuring instrument 51: Display panel showing the measurement results of the ammonia meter.

Claims

1. A method for quantifying the blood phenylalanine concentration of a subject using a test piece consisting of at least two sheet-like members, The test specimen comprises a first sheet-like member having a sample-holding layer containing an alkaline buffer and a second sheet-like member having an indicator layer that changes color upon reaction with ammonia gas, which are laminated together in a peeled or peelable state. The aforementioned quantitative method is a method comprising the following steps (A) to (D): Method for quantifying phenylalanine in blood: (A) A step of dropping blood (test blood) collected from a subject, either as is or after dilution, onto the sample holding layer of the first sheet-like member of the test piece, while separated from the second sheet-like member, and leaving it for a certain period of time. (B) Laminating the first sheet-like member onto the second sheet-like member such that the sample-holding layer of the first sheet-like member and the indicator layer of the second sheet-like member overlap in a non-contact state, dropping a phenylalanine ammonia lyase-containing solution onto the sample-holding layer, and leaving it for a certain period of time. (C) A step of calculating the ammonia concentration in the test blood from the degree of color of the indicator layer of the second sheet-like member, and (D) A step of calculating the blood phenylalanine concentration of the subject from the ammonia concentration.

2. The alkali buffer in the sample holding layer of the first sheet-like member is an alkali buffer containing boric acid and sodium hydroxide. The method for quantifying blood phenylalanine according to claim 1, wherein the indicator used in the indicator layer of the second sheet-like member is bromocresol green.

3. The content of boric acid and sodium hydroxide in the sample holding layer of the first sheet-like member is 0.40 to 0.45 mg and 0.15 to 0.20 mg per member, respectively. The bromocresol green content in the indicator layer of the second sheet-like member is 0.3 to 0.5 mg per member. In step (A) above, the amount of test blood applied to the sample holding layer of the first sheet-like member is at least 10 μl. The amount of phenylalanine ammonia lyase in the phenylalanine ammonia lyase-containing solution dropped onto the sample holding layer in step (B) is at least 2 mU. The method for determining blood phenylalanine according to claim 2, wherein the blood phenylalanine concentration that can be calculated in step (D) is 0 to 1400 μM.

4. The method for quantifying phenylalanine in blood according to claim 3, wherein step (C) is a step of measuring the absorbance of the color of the indicator layer at a wavelength of 635 nm.

5. The method for quantifying phenylalanine in blood according to claim 1, wherein the blood (test blood) collected from the subject is capillary blood.

6. The method for quantifying phenylalanine in the blood according to claim 1, wherein the subject is a patient with phenylketonuria.

7. A kit containing at least (a) and (b), or (a) to (c), for use in the method for quantifying blood phenylalanine as described in any one of claims 1 to 6: (a) A test specimen comprising a first sheet-like member having a sample-holding layer containing an alkaline buffer and a second sheet-like member having an indicator layer that changes color upon reaction with ammonia gas, laminated in a peeled or peelable state, (b) Phenylalanine ammonia lyase, (c) Solvent.

8. A method for managing blood phenylalanine levels in patients with phenylketonuria, The control method, comprising periodically or irregularly performing the method for quantifying the blood phenylalanine concentration described in any one of claims 1 to 6, using capillary blood as a test sample.

9. The control method according to claim 8, wherein the method for quantifying the blood phenylalanine concentration is carried out using the kit described in claim 7.

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