JPH1048193A - Testpiece for measuring magnesium in humor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply measure magnesium in a humor such as blood serum, blood plasma, etc., by using a testpiece including a specific component such as o-cresol phthalein complexon or the like as a reagent component. SOLUTION: O-cresolphthalein complexon is used as a metallic chelate indicator component, and O,O'-bis(2-aminophenyl)ethylene glycol-N,N,N',N'-tetraacetate is used as a calcium-masking agent. A pH buffer agent buffering to pH8.5-11.0 is also used, thereby obtaining a reagnet component. The test element for measuring magnesium in a humor is produced in this manner. The dry testpiece shows a low background value, good measuring accuracy in the whole of a wide measuring range and good storage stability without being influenced by calcium at all. When the reagent component is included in a binder layer of the testpiece, generated color can be uniform at the measuring time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a dry test strip for easily measuring magnesium in a liquid sample, particularly in a body fluid such as serum or plasma.

[0002]

2. Description of the Related Art In vivo, magnesium is a divalent metal having the second highest content after calcium, and is an important essential element deeply involved in various reactions in the living body. The concentration of magnesium in serum and plasma fluctuates antagonistically with the concentration of calcium, and the fluctuations are observed during dysfunction of the central nervous system and heart, or during renal failure and acute pancreatitis.
Magnesium measurement is becoming an important test item.

Various methods for colorimetrically measuring and measuring the magnesium concentration in a liquid sample using a divalent metal chelate indicator have been known. The most utilized metal chelate indicator is xylidyl blue-I. According to the 19th edition of Dojindo Chemical Catalog (issued on May 31, 1994), Eriochrome Black T, Carcyclome,
It is described that carboxyarsenazo, carmagite, chlorophosphonazo III, methylthymol blue, O-cresolphthalein complexone, spans, xylenol orange, and the like can also perform colorimetric determination of magnesium.

[0004] In the above metal chelate indicators used in the prior art, in all the indicators except formazan derivatives, the chelating function part has an allylazo structure and the dye part has a naphthol structure.

[0005] When the metal chelate indicators are used for colorimetric determination of magnesium in a biological sample, a masking agent for calcium is added in order to avoid the influence of coexisting calcium, regardless of which indicator is used. There is a need.

As a selective masking agent for calcium in the coexistence of magnesium and calcium, O, O'-bis (2-aminomethyl) ethylene glycol-N, N,
N ', N'-tetraacetic acid (abbreviated as GEDTA) is often used, and other examples include trans-1,2-diaminocyclohexane-N, N, N', N ',-tetraacetic acid (abbreviated as C).
yDTA), O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N ', N'-tetraacetic acid (abbreviation: BAPTA) and the like are known.

It is also known to add triethanolamine or a cyanide compound to mask other divalent metals, for example, iron, copper and zinc.

Preparation of a reagent component for measuring magnesium by a combination of these can be easily conceived by those skilled in the art, and many reagent components for measuring magnesium have been devised and implemented. For example, the most commonly used combination is xylidyl blue I and GED
This is a combination of TAs, and many reagent kits are commercially available. In the patent, chlorophosphonazo III and GEDT
A or combination of BAPTA (Japanese Unexamined Patent Publication No.
No. 64), Arsenazo I and GEDTA or BAPTA
(EP 597900), a method using a formazan derivative (JP-A-5-11908), and the like.

Also, US Pat. No. 5,397,710 relates to a dry test strip for whole blood having a blood cell separating ability, and the combination disclosed therein is such that the indicator is eriochrome black T, carmagite, magon, chlorophosphonazo II.
I, hydroxynaphthol blue, arsenazo I, spans and the like, and GEDTA is used as a masking agent.

[0010]

[0005] Drying reagents such as dry test strips are widely used because they are faster, simpler and cheaper than conventional liquid reagents in clinical diagnosis. When using a test strip to clinically diagnose a target substance in a liquid sample, the sample is often undiluted serum, plasma, or urine, etc., and the analyte is contained at a considerably high concentration. There are many. The case of magnesium is no exception, and in the case of serum and plasma, the human normal value is 1.8 ~.
2.0 mg / dl, 5.0-6.0.
It can reach 0 mg / dl. The test piece must be capable of measuring these outliers.

That is, when a dry test piece for measuring magnesium having a performance capable of measuring an abnormal value is prepared using a metal chelate indicator, the magnesium concentration in the sample (the target concentration of the upper limit of the measurement range including the abnormal value) ), It is necessary to include a necessary and sufficient amount of a chelate indicator as a reagent component in advance.

However, it has been found that when the above-mentioned chelating indicator is added in such a necessary and sufficient amount, considerable coloring is observed even in a free and endless state in which no complex is formed with magnesium. That is, since the background of the reagent is colored intensely at the initial value (hereinafter referred to as a high background value), there is a disadvantage that the measurement accuracy of the low-concentration magnesium sample is deteriorated.

The magnesium concentration of a biological sample, particularly serum or plasma, is extremely homeostatic, and the normal value is limited to a very narrow range. In diagnosing a disease, it is important to detect a slight deviation from a narrow range, and thus poor measurement accuracy is a fatal drawback.

Further, the present inventors dry-processed a combination of a conventional magnesium chelate indicator and a conceivable reagent component of a calcium masking agent to produce various magnesium measurement test pieces. It has been found that all combinations, including combinations, are necessarily affected by calcium.

[0015] The storage stability of the dry test specimens thus obtained was often unstable even under refrigerated storage conditions (under 4 to 8 degrees Celsius).

Accordingly, an object of the present invention is to provide a dry-type magnesium measurement test which has a low background value, has good measurement accuracy over a wide measurement range, is not affected by calcium, and has good storage stability. Is to provide a piece.

[0017]

The present inventors have used o-cresolphthalein complexone (abbreviated as OCPC) as a metal chelate indicator component and O, O'-bis (2-amino) as a calcium masking agent. Phenyl) ethylene glycol-N, N, N ', N'-tetraacetic acid (abbreviated name: BAPTA), and a pH buffer used to buffer the pH to 8.5 to 11.0. By doing so, it has been found that a test piece for measuring magnesium capable of solving all of the above problems can be produced.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A technique for measuring calcium or magnesium using OCPC is already known, but there is no technique applied to specific measurement of magnesium by selectively combining OCPC and BAPTA. It is a thing. The present inventors have prepared a test piece using OCPC as a metal chelate indicator component as a reagent component and BAPTA as a masking agent for calcium, and completely reduce the influence of calcium without lowering the sensitivity of magnesium measurement. I found that I could shut it off.

Since OCPC and BAPTA are both chelating agents for divalent metals, they react with both magnesium and calcium. However, OCPC chelate far more strongly than BAPTA for magnesium, and B
APTA chelate much more strongly. The present invention has been completed by applying this principle and specifically selecting a combination of OCPC and BAPTA.

Since the color of the reagent portion of the dry test piece is optically measured, it is naturally preferable that the background color of the reagent portion is low, but the concentration of the chelate indicator is insufficient. In the case of, measurement near the upper limit of the measurement range becomes impossible, so it is necessary to add to a high concentration. However, as described above, if the amount is excessively added, the background value of the reagent portion increases. However, the OCPC used in the present invention has a very low background value, even when added at a high concentration or compared with other chelating indicators, and is pale before reaction even when added in large amounts to make a test piece. It is pink and almost colorless. Therefore, the measurement precision is maintained and a test piece with high precision can be completed.

The sensitivity at the time of measuring magnesium by OCPC increases as the pH of the reaction system increases (as the pH increases). However, OCPC develops color on the alkaline side even without magnesium.
In other words, when processing into dry test specimens, the background value increases when the alkaline side is set to increase the sensitivity to increase the sensitivity, and the sensitivity decreases when near neutral,
For this reason, practical use has been postponed until now. However, in the present invention, the pH of the reaction system is adjusted to pH 8.5 to 11.0.
By using a buffer, a test piece with a low background was obtained without a decrease in sensitivity. This is due to the influence of carbon dioxide contained in the atmosphere during the preparation of the test piece.
The decrease in H is also suppressed.

As long as the pH buffer can be used in the range of pH 8.5 to 11.0 and does not volatilize upon drying, any of buffers known and used in this type of test strip can be used. Can be used with Specific examples include boric acid-sodium hydroxide buffer, sodium bicarbonate-sodium carbonate buffer, glycine-sodium hydroxide buffer, CHE
S buffer, CAPSO buffer and the like. pH 8.5-
If it is in the range of 11.0, the measurement of magnesium is sufficiently possible, but more preferably in the range of 9.5 to 10.0.

In a general metal chelate reaction system including OCPC, when measuring magnesium in a body fluid, particularly a serum / plasma sample, it is also known that the measurement is affected by proteins in the sample, particularly albumin. This is due to the property that magnesium is adsorbed on albumin,
Of the magnesium in serum and plasma, the ratio of magnesium adsorbed on albumin is almost constant at about 30%, and is quantified during measurement and easily canceled, so that there is usually no practical problem.

However, in the case of a sample having an extremely high albumin concentration, which may occur in rare cases, the effect may not be negligible. In the present invention, by further adding an ionic surfactant, it has become possible to suppress and avoid variations among a plurality of samples including samples having extremely high albumin concentrations. Specific examples of the ionic surfactant include sodium dodecyl sulfate for the anionic surfactant and tetradecyltrimethylammonium bromide for the cationic surfactant, and any ionic surfactant having the same action can be used. It will be apparent to one skilled in the art that compounds may be used.

Among the test strips for measuring magnesium containing the reagent component of the present invention, a form which can be preferably applied is formed of a detection site containing the reagent component of the present invention and a water-impermeable support serving as a base for the detection site. This is a general test piece. The detection site is composed of a water-soluble binder layer laminated on a support and a sample holding layer made of a water-insoluble porous material. The reagent component of the present invention is present on only one layer of the binder layer or the sample holding layer or on both layers.

It is preferable that OCPC is contained in a range of 0.05 to 0.3% by dry weight% with respect to the whole detection site.

BAP for masking calcium
TA may be added in a sufficient amount with respect to the concentration of calcium expected to coexist. When measuring serum / plasma, it is preferable that BAPTA is contained in a range of 0.3 to 5.0% by dry weight% with respect to the entire detection site.

The pH buffer for buffering to the alkaline side is preferably 0.3 to 5.0% by dry weight% in the whole detection site.

When the binder layer contains a reagent component, the binder layer serves as an adhesive for laminating the sample holding layer at the time of production of the detection site, and at the same time, allows the reagent component to be present evenly and uniformly. This is a layer for keeping color development uniform.

Since the sample is an aqueous liquid such as serum or plasma, the binder layer is made of a water-soluble polymer, and any material can be used as long as it does not substantially contain magnesium or calcium. Specific examples include polyacrylamide and polyvinylpyrrolidone.
These polymers are added to an aqueous solution containing a reagent component, and the aqueous solution is adjusted to have an appropriate viscosity to obtain a coating solution.
The concentration in the state of a coating solution is 4.0 to 6.0% by weight for polyacrylamide, and 10 to 15% by weight for polyvinylpyrrolidone. The amount of the polymer used to adjust the viscosity to an appropriate level greatly varies within the range of 0.5 to 20% by weight% after drying depending on the type of the polymer, based on the entire detection site. The above-mentioned binder layer does not contain a reagent component, and the reagent component can be present in an impregnated form only in the sample holding layer shown below.

When the sample is applied, the sample holding layer uniformly spreads the sample laterally on the surface of the detection site, reacts the reagent with the sample while holding the sample, and is ready for optically detecting the reagent after the reaction. It is used for the purpose of As the insoluble porous material used for the sample holding layer, a filter paper made of cellulose or glass fiber, a membrane filter, a woven fabric made of natural fibers or chemical fibers, etc. are used after washing and drying as necessary. As a preferred specific example, a woven fabric is used and is pressed and laminated on the binder layer in a wet state or a dry state. Dry weight% of water-insoluble porous material used as sample holding layer
Occupies 65 to 95% of the entire detection site.

The water-impermeable support used as a base for the detection site is made of a plastic such as polyethylene terephthalate or polystyrene used in this type of dry test piece and has a thickness of 50 μm to 1 mm, preferably 100 μm.
A white film of μm to 300 μm having a roughened surface is used.

The coating solution serving as the base of the binder layer is uniformly applied on a water-impermeable support in a wet thickness range of 50 μm to 300 μm. Preferably wet thickness 100 to 200
The range of μm is good, and a known method may be used as a coating method. The applied binder layer is blown dry with warm air at 40 to 60 ° C.

When the sample holding layer is laminated on the binder layer, the reagent components in the binder layer may move together with the polymer to the sample holding layer. At the detection site defined in the present invention, both are clearly separated. You may not. In any case, both the sample holding layer and the binder layer in the detection site optionally contain the combination of OCPC and BAPTA used in the present invention and a pH buffer.

When an ionic surfactant is added to a high-concentration albumin sample, the amount of the ionic surfactant in the entire detection site is preferably 0.3 to 20% by dry weight%. Up to 1.0% by dry weight of surfactant addition,
The effect of avoiding the effect of albumin appears clearly, but from around 1.0%, the effect of avoiding the effect does not appear so large with respect to the amount of the surfactant added. However, the more it is added, the more effective it is, until it is saturated (dry weight%
20%) can be added.

Other components such as a cyanide compound for masking metals other than calcium and a stabilizer for a metal chelate indicator can be added to the detection site.

The detection site raw material of the dry test specimen of the present invention obtained as described above is cut into small pieces together with a water-impermeable support, and the water-impermeable support is placed on a strip-shaped base film for holding with a finger. Paste with the body side on the film side to complete as a test piece that is easy to hold. FIG. 1 shows a cross-sectional view of this state. This base film is merely a handle and is not an essential component of the present invention. If the raw material of the detection site is cut into a strip shape as it is together with the impermeable support, the test piece according to the present invention can be obtained without a base film.

An appropriate amount of the liquid sample is spotted on the detection site of the test piece, and after the reaction is completed, the reflectance is measured from the detection site side or the support side. The body and handle material must be light transmissive). The wavelength should just be a wavelength near the absorption peak of the chelate complex of magnesium and OCPC. Preferably 5
65-575 nm.

[0040]

EXAMPLES The procedure for preparing a dry test piece for measuring magnesium containing the reagent component of the present invention and the measurement results using the obtained test piece are shown below.

Preparation of Reagent Component Solution (Coating Solution) (Prescription) o-Cresolphthalein complexone (Dojindo); 1.50 mmol / L O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N ′, N′-tetraacetic acid tetrapotassium salt (Dojindo); 16 mmol. L sodium borate buffer (pH 9.5) (Wako Pure Chemical Industries);
300 mmol / L sodium dodecyl sulfate (Wako Pure Chemical Industries); 10.0% by weight 10% polyacrylamide (Nacalai Tesque); 5.0
weight%

Formation of raw material at detection site The above solution was coated on a 200 μm thick white polyethylene terephthalate film to a thickness of 120 μm (wet state).
And dried with air at 45 ° C. for 15 minutes. to this,
A woven fabric having a thickness of about 250 μm uniformly moistened with an aqueous solution of 0.1% by weight Triton X-100 (Nacalai Tesque) was pressure-bonded, and air-dried again at 45 ° C. for 15 minutes.

The raw material of the detection site after drying contains the following components per 1 m ² . o-cresolphthalein complexone; 0.09% O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N ', N'-tetraacetic acid tetrapotassium salt; 0.88% boric acid; 1.80% sodium hydroxide; 0.78% sodium dodecyl sulfate; 10.86% polyacrylamide; 0.71% Triton X-100; 0.03% woven fabric; 84.86%

Preparation of Test Piece The obtained test piece raw material was cut into small pieces of 5 mm × 7 mm.
Strip-shaped (5 mm x 80 mm) thickness 0.5 to be used as a handle
A double-sided tape was attached to the tip of a base film made of white polyethylene terephthalate (mm) to obtain a test piece. A cross-sectional view near the detection site at this time is the same as that in FIG.

● Measuring operation As a measuring device, a desktop reflectance measuring device, Spotchem SP-4410, manufactured by Kyoto Daiichi Kagaku Co., Ltd. was used. Place the test piece on a table whose temperature has been adjusted to 37 ° C.
0 Aμl of serum sample was spotted. 575n wavelength after 3 minutes
The reflectance (R) at m was measured. The reflectance (R) was converted to a K / S value by the Kubelka-Munk equation (*). * Kubelka-Munk formula: K / S = (1-R) ² / 2R

By measuring a sample having a known concentration, “K
A calibration curve between “/ S value−magnesium concentration” was prepared in advance. The calibration curve is shown in FIG. Based on this, the magnesium concentration in the sample was calculated.

Confirmation of Simultaneous Reproducibility In order to show the simultaneous reproducibility of the test piece of the present invention, three types of serum of a low concentration (sample 1), a normal concentration (sample 2) and a high concentration (sample 3) of magnesium were used. Table 1 shows the results of simultaneous measurement of each sample 12 times. Each sample showed very good simultaneous reproducibility with a coefficient of variation within 3%.

Confirmation of Effect of Calcium In order to show that the test piece of the present invention is not affected by calcium, calcium chloride was added to normal serum serum and high-concentration serum, and the influence on the measured value of magnesium was examined. Was. As a result, as shown in FIG. 3, it was found that there was no effect at all up to a calcium addition concentration of 20 mg / dl.

Confirmation of storage stability The test piece of the present invention was sealed in a glass bottle together with a desiccant,
The stability when stored in a refrigerated state (4 ° C.) was examined. After the number of days (initial, 6, 12, 18 months) indicated on the horizontal axis of FIG. 4, the normal magnesium serum and the high-concentration serum were measured 6 times each using the preserved test piece. When a graph was drawn by averaging six times, almost no change in sensitivity was observed as shown in FIG. 4, and it was confirmed that the test piece was a very stable test piece.

[0050]

As described in detail above, according to the present invention,
Unlike conventional test strips containing reagent components, it has good measurement accuracy over the entire measurement range, low background value,
It was possible to obtain a dry test piece for measuring magnesium in a body fluid in a short time, which is not affected by any coexisting calcium, has good storage stability, and is very easy to use.

[0051]

[Brief description of the drawings]

Table 1 is a table showing the results of the simultaneous reproducibility of the test pieces obtained by the present invention. FIG. 1 is a cross-sectional view of the vicinity of a detection site of a test piece of the present invention. FIG. 2 is a calibration curve when actually performing optical measurement using the test piece of the present invention. FIG. 3 is a graph showing the effect of calcium on the test piece of the present invention. FIG.
3 is a graph showing the storage stability of the test piece of the present invention.

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[Procedure amendment]

[Submission date] July 12, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0047

[Correction method] Change

[Correction contents]

Confirmation of Simultaneous Reproducibility In order to show the simultaneous reproducibility of the test piece of the present invention, three types of serum of a low concentration (sample 1), a normal concentration (sample 2) and a high concentration (sample 3) of magnesium were used. Table 1 shows the results of simultaneous measurement of each sample 12 times. Each sample showed very good simultaneous reproducibility with a coefficient of variation within 3%.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of the vicinity of a detection site of a test piece of the present invention.

FIG. 2 is a graph showing a calibration curve when actually performing optical measurement using the test piece of the present invention.

FIG. 3 is a graph showing the effect of calcium on the test piece of the present invention.

FIG. 4 is a graph showing the storage stability of the test piece of the present invention.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hisashi Ashida 57 Kyoto Higashikujo Nishiakeda-cho, Minami-ku, Kyoto City, Kyoto Prefecture

Claims (6)

[Claims] 1. A test strip for measuring magnesium in a body fluid, comprising at least the following components as reagent components: o-Cresolphthalein complexone O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N ', N'-tetraacetic acid pH buffer for buffering pH 8.5 to 11.0 2. The method of claim 2, wherein the pH buffer is more preferably pH
2. The test piece according to claim 1, wherein the test piece is buffered to 9.5 to 10.0. 3. The composition further comprises an ionic surfactant.
The test piece according to claim 1. 4. A test piece comprising a water-impermeable support and a detection site carried on the support, wherein the detection site comprises:
A sample holding layer comprising a water-insoluble porous material, and a binder layer comprising a water-soluble polymer, wherein the reagent component according to claim 1 is provided on only one of the sample holding layer and the binder layer or on both layers. A test piece for measuring magnesium in a body fluid, wherein the test piece is made to exist. 5. The composition further comprising an ionic surfactant.
The test piece according to claim 4. 6. The test strip according to claim 5, wherein a dry weight% ratio of each component at the detection site is as follows. O-cresolphthalein complexone; 0.05
-0.3% O, O'-bis (2-aminophenyl) ethylene glycol-N, N, N ', N'-tetraacetic acid; 03-5.0 pH buffer; 03-5.0 Ionic surfactant; 03-20% Water-soluble polymer; 0.5-20 Water-insoluble porous material; 65-95%