JP3809484B2 - Test piece for measuring magnesium in body fluids - Google Patents

Description

【００４８】
●カルシウムの影響の確認
本発明の試験片においてカルシウムの影響を受けないことを示すために、塩化カルシウムをマグネシウム正常値血清及び高濃度血清へ添加し、マグネシウム測定値へ与える影響を調べた。結果、図３に示すとおりに、カルシウム添加濃度２０ｍｇ／ｄｌまで全く影響を受けないことが分かった。
【００４９】
●保存安定性の確認
本発明の試験片を乾燥剤とともにガラス瓶中に密封し、冷蔵状態（４℃）で保存した場合の安定性を調べた。図４の横軸に示した日数（初期，６，１２，１８ヶ月）経過した後に、保存後の試験片を利用してマグネシウム正常値血清と高濃度血清を各６回ずつ測定した。６回の平均を取りグラフを描いたところ、図４に示したとおり感度の変化はほとんど認められず、非常に安定な試験片であることが確認された。
【００５０】
【発明の効果】
以上詳述したように、本発明によれば、従来の試薬成分を含む試験片と異なり、測定レンジ全域で良好な測定精度を有し、バックグラウンド値が低く、共存するカルシウムの影響も一切受けない、保存安定性が良い、使用が非常に簡便な、体液中のマグネシウムを短時間に測定するための乾式試験片を得ることができた。
【００５１】
【図面の簡単な説明】
【図１】図１は、本発明の試験片の検出部位付近の断面図である。
【図２】図２は、本発明の試験片で実際に光学測定する際の検量線を示したグラフである。
【図３】図３は、本発明の試験片に対するカルシウムの影響を示したグラフである。
【図４】図４は、本発明の試験片の保存安定性を示したグラフである。[0001]
[Industrial application fields]
The present invention relates to a dry test piece for easily measuring magnesium in a liquid sample, particularly body fluid such as serum and plasma.
[0002]
[Prior art]
In the living body, magnesium is a divalent metal having the second largest content after calcium and is an important essential element deeply involved in various in vivo reactions. Magnesium concentration in serum and plasma fluctuates antagonistically with calcium concentration, and is observed during central nervous system and heart dysfunction, renal failure, acute pancreatitis, etc., so measurement of magnesium is important It is becoming one of the important inspection items.
[0003]
Various methods for colorimetric determination and measurement of 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 Dojin Chemical Catalog 19th edition (issued on May 31, 1994), Eriochrome Black T, Calcyclome, Carboxyarsenazo, Carmagite, Chlorophosphonazo III, Methylthymol Blue, O-Cresol It is described that phthalein complexone, spans, xylenol orange and the like can also be used for colorimetric determination of magnesium.
[0004]
In the metal chelate indicators used in the above-mentioned prior art, in any of the indicators except formazan derivatives, the chelating function portion has an allylazo structure, and the dye portion has a naphthol structure.
[0005]
When colorimetrically quantifying magnesium in biological samples with these metal chelate indicators, it is necessary to add a masking agent for calcium in order to avoid the influence of coexisting calcium, regardless of which indicator is used. .
[0006]
As a selective masking agent for calcium in the presence of magnesium and calcium, O, O′-bis (2-aminomethyl) ethylene glycol-N, N, N ′, N′-tetraacetic acid (abbreviated as GEDTA) is available. Other often used are trans-1,2-diaminocyclohexane-N, N, N ′, N ′,-tetraacetic acid (abbreviation CyDTA), O, O′-bis (2-aminophenyl) ethylene Glycol-N, N, N ′, N′-tetraacetic acid (abbreviation BAPTA) and the like are known.
[0007]
A method of adding triethanolamine or a cyanide compound to mask other divalent metals such as iron, copper and zinc is also known.
[0008]
Preparation of magnesium measurement reagent components by these combinations can be easily considered by those skilled in the art, and many magnesium measurement reagent components have been devised and implemented. For example, the most widely used combination is a combination of xylidyl blue I and GEDTA, and many reagent kits are commercially available. Patents include a combination of chlorophosphonazo III and GEDTA or BAPTA (Japanese Patent Laid-Open No. 4-120464), a combination of arsenazo I and GEDTA or BAPTA (European Patent 597900), and a method using a formazan derivative (Japanese Patent Laid-Open No. 5-111908). ) Etc. are disclosed.
[0009]
U.S. Pat. No. 5,397,710 relates to a dry test piece for whole blood having blood cell separation ability, and the disclosed combination is that the indicator is Eriochrome Black T, Karmagite, Magone, Chlorophosphonazo III, Hydroxynaphthol Blue , Arsenazo I, Spans, etc., and GEDTA is used as the masking agent.
[0010]
[Problems to be solved by the invention]
Now, dry reagents such as dry test strips are frequently used in clinical diagnosis because they are quicker, simpler, and less expensive than conventional liquid reagents. When clinically diagnosing a target substance in a liquid sample using a test piece, the sample is often undiluted serum, plasma, urine, etc., and the target substance is contained in a considerably high concentration There are many. In the case of magnesium, there is no exception, and in the case of serum / plasma, even human normal values may reach 1.8 to 2.0 mg / dl, and in the case of abnormally high concentrations, it may reach 5.0 to 6.0 mg / dl. The specimen must be capable of measuring these outliers.
[0011]
In other words, when making a magnesium test dry test piece that has the ability to measure outliers using a metal chelate indicator, relative to the magnesium concentration in the sample (target concentration range upper limit concentration including outliers) Therefore, it is necessary to include a necessary and sufficient amount of chelate indicator in advance as a reagent component.
[0012]
However, it has been found that when the above-mentioned chelate indicator is added in such a necessary and sufficient amount, considerable coloring is observed even in a free and end-colored state where no complex is formed with magnesium. That is, since the initial value shows a deep coloration of the reagent background (hereinafter referred to as a high background value), the measurement accuracy of the magnesium low-concentration sample is deteriorated.
[0013]
The magnesium concentration of biological samples, particularly serum and plasma, is extremely homeostatic, and normal values are limited to a very narrow range. In the diagnosis of a disease, since it is important to detect a slight deviation from the narrow range, the poor measurement accuracy is a fatal defect.
[0014]
Furthermore, the present inventors dried various combinations of conventional reagent components of magnesium chelate indicator and calcium masking agent to produce various magnesium measurement specimens, including the same combinations as in the prior art. All combinations have been found to be affected by calcium.
[0015]
And also about the storage stability of the dry-type test piece obtained in that way, there were many things which were unstable also in the refrigerated storage state (4-8 degree Celsius conditions).
[0016]
Therefore, the object of the present invention is to provide a dry magnesium measurement specimen that has a low background value, has good measurement accuracy over a wide measurement range, is not affected by calcium, and has good storage stability. It is to be.
[0017]
[Means for Solving the Problems]
The present inventors use o-cresolphthalein complexone (abbreviated name OCPC) as a metal chelate indicator component, and O, O′-bis (2-aminophenyl) ethylene glycol-N, N, as a calcium masking agent. By using N ′, N′-tetraacetic acid (abbreviated name BAPTA) and further using a pH buffering agent buffering to pH 8.5 to 11.0, the above-mentioned problem can be obtained by using it as a reagent component of a dry test piece. It was discovered that magnesium test specimens that can be solved all can be produced.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
A technique for measuring calcium or magnesium using OCPC is a known method, but there has been no technique applied to specific measurement of magnesium by selectively combining OCPC and BAPTA. When the present inventors prepared a test piece using OCPC as a metal chelate indicator component as a reagent component and BAPTA as a masking agent for calcium, the influence of calcium was completely eliminated without lowering the sensitivity of magnesium measurement. I found out that it can be blocked.
[0019]
Since OCPC and BAPTA are both chelating agents for divalent metals, they react with both magnesium and calcium. However, OCPC is much more chelated to magnesium than BAPTA, and BAPTA is much more chelated to calcium than OCPC. The present invention has been completed by applying this principle and specifically selecting a combination of OCPC and BAPTA.
[0020]
Since the color of the reagent portion of the dry test piece is optically measured, it is natural that the background portion of the reagent portion should have a low degree of coloration, while the chelating indicator concentration is insufficient. Since measurement near the upper limit of the measurement range becomes impossible, it must be added to a high concentration. However, as described above, if the amount is excessively added, the background value of the reagent portion becomes high. However, the OCPC used in the present invention has a very low background value even when added to a high concentration or compared to other chelate indicators, and even if added in a large amount to form a test piece, it is light before the reaction. It is pink and nearly colorless. Therefore, measurement accuracy is maintained and a test piece with high precision can be completed.
[0021]
The sensitivity at the time of magnesium measurement by OCPC increases as the pH of the reaction system increases (the alkalinity increases). However, OCPC develops color when it is on the alkaline side even if magnesium is not present. In other words, when processing into a dry test piece, the background value becomes high if it is made strongly alkaline to increase the sensitivity, and the sensitivity decreases if it is close to neutral, so that it has not been put to practical use until now. It was. However, in the present invention, the use of a pH buffer that buffers the reaction system to pH 8.5 to 11.0 yielded a test piece with a low background without a decrease in sensitivity. At the same time, a decrease in pH due to the influence of carbon dioxide contained in the atmosphere during the preparation of the test piece is also suppressed.
[0022]
As long as the pH buffer can be used in the range of pH 8.5 to 11.0 and the components do not volatilize even when dried, this kind of test piece can be used optionally from among publicly known buffers. . Specific examples include boric acid-sodium hydroxide buffer, sodium bicarbonate-sodium carbonate buffer, glycine-sodium hydroxide buffer, CHES buffer, CAPSO buffer, and the like. If the pH is in the range of 8.5 to 11.0, magnesium can be measured sufficiently, but more preferably in the range of 9.5 to 10.0.
[0023]
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 sample is affected by proteins in the sample, particularly albumin. This is due to the property that magnesium is adsorbed to albumin, but the ratio of magnesium adsorbed to albumin is approximately 30% of the serum / plasma magnesium, and it is easy to quantify during measurement. Since it is canceled, there is usually no problem in practice.
[0024]
However, in the case of a sample with extremely high albumin concentration that may occur rarely, there are cases where the influence cannot be ignored. In the present invention, by further adding an ionic surfactant, it is possible to suppress and avoid variations among a plurality of specimens including a sample having an extremely high albumin concentration value. Specific examples of ionic surfactants include sodium dodecyl sulfate for anionic surfactants and tetradecyltrimethylammonium bromide for cationic systems. Any ionic surfactant having the same action can be used. It will be apparent to those skilled in the art that the compounds may be used.
[0025]
Of the test pieces for measuring magnesium containing the reagent component of the present invention, a form that can be preferably applied is formed of a detection site containing the reagent component of the present invention and a water-impermeable support that is the foundation of the detection site. It 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 only in one layer of the binder layer or the sample holding layer or across both layers.
[0026]
It is preferable that OCPC is contained in the range of 0.05 to 0.3% by dry weight% with respect to the entire detection site.
[0027]
A sufficient amount of BAPTA for masking calcium may be added to the concentration of calcium expected to coexist. In the case of 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.
[0029]
The pH buffer that buffers the alkaline side is preferably 0.3 to 5.0% by dry weight in the entire detection site.
[0030]
When the binder layer contains a reagent component, the binder layer becomes an adhesive for laminating the sample holding layer during the production of the detection site, and at the same time, the reagent component is uniformly present and the color development is uniform during measurement. It is a layer for keeping it.
[0031]
Since the sample is an aqueous liquid such as serum or plasma, a water-soluble polymer is used as the material for the binder layer, and any material that does not substantially contain magnesium or calcium can be used. Specific examples include polyacrylamide and polyvinylpyrrolidone. These polymers are added to an aqueous solution containing reagent components, and the aqueous solution is prepared to have an appropriate viscosity to obtain a coating solution. The concentration in the coating solution state is 4.0 to 6.0% by weight for polyacrylamide and 10 to 15% by weight for polyvinylpyrrolidone. The amount of the polymer used for adjusting to an appropriate viscosity varies greatly within the range of 0.5 to 20% in terms of the weight% after drying depending on the type of the whole detection site. The 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.
[0032]
The purpose of the sample holding layer is to spread the sample evenly on the surface of the detection site when the sample is applied, to allow the reagent to react with the sample while holding the sample, so that the reagent after the reaction can be optically detected It is used in. 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 fiber or chemical fiber, and the like are washed and dried 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. The dry weight of the water-insoluble porous material that serves as the sample holding layer accounts for 65 to 95% of the entire detection site.
[0033]
The water-impermeable support used as the base of 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 to 300 μm. A film whose surface is roughened is used.
[0034]
The coating liquid that is the basis of the binder layer is uniformly applied on a water-impermeable support in a wet thickness range of 50 μm to 300 μm. The wet thickness is preferably in the range of 100 to 200 μm, and a known method may be used as the coating method. The applied binder layer is blown and dried with hot air of 40 to 60 ° C.
[0035]
When laminating the sample holding layer on the binder layer, the reagent component in the binder layer may move to the sample holding layer together with the polymer, and the two are not clearly separated at the detection site defined in the present invention. There may be cases. In any case, both the sample holding layer and the binder layer in the detection site optionally contain a combination of OCPC and BAPTA used in the present invention and a pH buffer.
[0036]
When the ionic surfactant is added to the high-concentration albumin sample, the amount of the ionic surfactant in the entire detection site is preferably 0.3 to 20% in terms of dry weight. When the addition of the surfactant is 1.0% by dry weight, the effect of avoiding the influence of albumin appears clearly, but from the point when it exceeds 1.0%, the effect of avoiding the amount of addition of the surfactant Does not appear so large. However, the more it is added, the more effective it is, so there is no problem even if it is added until it is saturated (20% by dry weight).
[0037]
Components other than these, for example, cyanide compounds for masking metals other than calcium, stabilizers for metal chelate indicators, and the like can be added to the detection site.
[0038]
The raw material of the dry test piece of the present invention obtained as described above is cut into small pieces together with a water-impermeable support, and the water-impermeable support side is filmed on a strip-shaped base film for holding with a finger. Affix to the side and complete as a test piece that is easy to hold. A sectional view of this state is shown in FIG. This base film is merely a handle and is not an essential component of the present invention. If the detection site raw fabric 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 the base film.
[0039]
Apply an appropriate amount of a liquid sample onto the detection site of the test piece, and measure the reflectance from the detection site side or the support side after the reaction is complete (however, when measuring from the support side, the support and handle) Must be light transmissive). The wavelength should just be a wavelength close | similar to the absorption peak of the chelate complex of magnesium and OCPC. Preferably it is 565-575 nm.
[0040]
【Example】
The production procedure of the magnesium test dry test piece containing the reagent component of the present invention and the measurement results using the obtained test piece are shown below.
[0041]
● Preparation (prescription) of reagent component solution (coating solution)
o-cresolphthalein complexone (Dojindo); 1.50 mmol / LO, 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% by weight
[0042]
Formation of detection sheet original material The above solution was applied on a white polyethylene terephthalate film having a thickness of 200 μm to a thickness of 120 μm (wet state), and air-dried at 45 ° C. for 15 minutes. 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 thereto, and air-dried again at 45 ° C. for 15 minutes.
[0043]
The detection site raw fabric 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%
[0044]
● Production of test piece The obtained test piece is cut into 5 mm x 7 mm pieces, and both sides are attached to the tip of a base film made of white polyethylene terephthalate with a strip shape (5 mm x 80 mm) and a thickness of 0.5 mm. A test piece was affixed with tape. A cross-sectional view of the vicinity of the detection site at this time is the same as FIG.
[0045]
Measurement Operation As a measuring device, a desk top reflectance measuring device Spot Chem SP-4410 of Kyoto Daiichi Kagaku Co., Ltd. was used. Place the test piece on a table whose temperature is adjusted to 37 ° C. A 0 μl serum sample was spotted. After 3 minutes, the reflectance (R) at a wavelength of 575 nm was measured. The reflectance (R) was converted to a K / S value by Kubelka-Munk's formula (*).
* Kubelka-Munk formula: K / S = (1-R) ² / 2R
[0046]
A calibration curve between “K / S value−magnesium concentration” was prepared in advance by measuring a sample having a known concentration. A calibration curve is shown in FIG. Based on this, the magnesium concentration in the sample was calculated.
[0047]
● Confirmation of simultaneous reproducibility In order to demonstrate the simultaneous reproducibility of the test piece of the present invention, each of three types of serum samples of low concentration (sample 1), normal concentration (sample 2), and high concentration (sample 3) of magnesium is shown. Table 1 shows the results of 12 simultaneous measurements. Each sample showed very good simultaneous reproducibility with a coefficient of variation of 3% or less.

[0048]
-Confirmation of the influence 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 and high-concentration serum to examine the influence on the magnesium measurement value. As a result, as shown in FIG. 3, it was found that the calcium addition concentration was not affected at all up to 20 mg / dl.
[0049]
-Confirmation of storage stability The test piece of the present invention was sealed in a glass bottle together with a desiccant, and the stability when stored in a refrigerated state (4 ° C) was examined. After the days (initial, 6, 12, 18 months) indicated on the horizontal axis of FIG. 4 elapsed, normal magnesium serum and high-concentration serum were measured 6 times each using the specimen after storage. When the average of 6 times was taken and the graph was drawn, the change of a sensitivity was hardly recognized as shown in FIG. 4, and it was confirmed that it is a very stable test piece.
[0050]
【The invention's effect】
As described above in detail, according to the present invention, unlike a test piece containing conventional reagent components, it has good measurement accuracy over the entire measurement range, has a low background value, and is not affected by coexisting calcium at all. A dry test piece for measuring magnesium in a body fluid in a short time was obtained.
[0051]
[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 optical measurement is actually performed with the test piece of the present invention.
FIG. 3 is a graph showing the influence 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.

Claims (6)

A test piece for measuring magnesium in body fluid, comprising at least the following components (1) to (3) as reagent components:
(1) o-cresolphthalein complexone
(2) O, O′-bis (2-aminophenyl) ethylene glycol-N, N, N ′, N′-tetraacetic acid
(3) pH buffer that buffers to pH 8.5 to 11.0   The test piece according to claim 1, wherein the pH buffer is more preferably buffered at pH 9.5 to 10.0.   Furthermore, the test piece of Claim 1 containing an ionic surfactant.   A test piece comprising a water-impermeable support and a detection site carried on the support, the detection site comprising a sample holding layer made of a water-insoluble porous material and a binder layer made of a water-soluble polymer A test piece for measuring magnesium in body fluid, characterized in that the reagent component according to claim 1 is present only in one layer of the sample holding layer and the binder layer or straddling both layers.   Furthermore, the test piece of Claim 4 containing an ionic surfactant. The test piece according to claim 5, wherein the 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; 0. 3 to 5.0 %
PH buffer; 0 . 3 to 5.0 %
An ionic surfactant; 0 . 3-20%
-Water-soluble polymer; 0.5-20 %
・ Water-insoluble porous material: 65-95%

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