JPH01129164A - Test piece for quantifying creatinine in body fluid and preparation thereof - Google Patents

Abstract

PURPOSE:To perform reaction on a test piece under a strong alkaline condition and to enhance preservability, by preparing the test piece for quantifying creatinine in the body fluid by utilizing a carboxylic acid derivative polymer. CONSTITUTION:A test piece for quantifying creatinine in the body fluid containing a reagent component reacting with creatinine at least under an alkaline condition to show a color, an alkali component and an alkali soluble carboxylic acid derivative polymer is prepared by successively immersing an absorbable carrier in an aqueous solvent containing at least the alkali component and the alkali soluble carboxylic acid derivative polymer and an org. solvent containing the reagent component reacting with creatinine at least under an alkaline condition to show a color and drying the impregnated carrier. The degree of coloration of the obtained test piece for quantifying creatinine is read by measuring reflectivity and compared with a preliminarily formed calibration curve to quantity the creatinine component.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a test piece for quantifying creatinine present in body fluids such as urine and serum, and a method for producing the same.

[Prior Art J Creatinine is a dehydrated product of creatine, which becomes creatine phosphate and is the final metabolite used as an energy source for muscle contraction. The generated creatinine is absorbed in the renal glomerulus and then excreted in the urine without being reabsorbed.

Quantification of creatinine in body fluids is useful for diagnosing severe muscle diseases or various renal diseases such as nephritis and renal failure.

Furthermore, it is known that the amount of creatinine excreted into the urine per day is not easily affected by the content of meals, amount of exercise, amount of urine, etc. Therefore, by quantifying the amount of creatinine in the as-needed method (urine collected only once at any time during the day), it is possible to estimate the proportion of the as-needed method to the total urine volume of the day. By utilizing these characteristics of urinary creatinine, it is possible to measure the total daily urinary excretion of proteins, electrolytes, hormones, drugs, etc., which requires complicated urine collection and imposes a large burden on the subject. can be estimated based on the sample.

Regarding the method for quantifying creatinine, U.S. Patent Nos. 3 and 5
Some prior art techniques are disclosed in US Pat. No. 57,018 and US Pat. No. 3,705,013. Among them, the so-called Jaffe method is used to colorimetrically quantify the red-yellow-orange creatinine picrate produced by the combination of alkaline picric acid and creatinine.
! jlshiz chemistry z(Z, Physol, Ghem,
) 41; 223, 1904] is still frequently used because it is easy to operate and has sufficient sensitivity.

In addition, Benedictobeur (Benedict-B) uses 3,5-dinitrobenzoic acid as a colorimetric method.
ehre ) Law I Journal Saibu Bio 0
J, Biol Chew,
114; 515, 1936], 1.4-naphthoquinone-2-sulfone method [Journal of
Bio ajihhga mistry (J, Bial・Ghe
233; 530, 1958] and the enzymatic method using creatinase
Iin, Chem,) 19;632゜1973
1, etc., but none of them are currently popular because they have problems in terms of sensitivity, operability, economy, etc. compared to the Jaffe method. For details on these methods for quantifying creatinine, please refer to page 57 of ``Complete Book of Clinical Laboratory Techniques, Clinical Chemistry Test UJC, edited by Noburu Ishii, published by Igakushoin, 1975.''

On the other hand, in recent years, a method of measuring body fluid production using a test piece in which reagent components are supported on an absorbent carrier such as T-paper or porous film has attracted attention due to its excellent reagent stability and operability. There is. The test piece for measuring creatinine is also an application of the Benediktobeur method [Clinical
Kamistryi (CIin, Che■・)Moe, 103
4.19811 and those using an enzymatic reaction system [Journal of the Japanese Society of Clinical Laboratory Automation, 5G, 1985, and Clinical Laboratory Instruments and Reagents, 115B, 19851 are known.

[Problems to be Solved by the Invention] The aforementioned test piece for measuring creatinine is commercially available for measuring blood creatinine concentration, but in principle it cannot be applied to measuring urinary creatinine. This is not possible.However, the urinary creatinine concentration can be several tens of times higher than the blood concentration, and in some cases even hundreds of times higher, and the urine p)I fluctuates greatly, unlike the blood pH, so one reaction is impossible. Dilution of urine is required prior to measurement to ensure a suitable pH for the system [CIin, Chem, 5; 1900, 1]
9851° One possible means of measuring creatinine without diluting the test urine is to mix an alkaline component into the reagent composition in advance to ensure a suitable pH for the reaction.

However, in reality, the alkaline components necessary for these reaction systems denature the absorbent carrier commonly used for test pieces.
It was difficult to manufacture a test piece for quantifying creatinine by impregnating it into an absorbent carrier.

[Means for Solving the Problem] The present invention enables application to an absorbent carrier for alkaline components. That is, the present invention provides a test piece for quantifying creatinine in body fluids, which is characterized by comprising at least a reagent component that reacts with alkaline creatinine to develop a color, an alkaline component, and an alkali-soluble carboxylic acid derivative polymer, and an absorbent Quantification of creatinine in body fluids, which comprises sequentially immersing a carrier in an aqueous solvent containing at least an alkaline component and an alkali-soluble carboxylic acid derivative polymer, and an organic solvent containing at least a reagent component that reacts with alkaline creatinine to develop a color, followed by drying. This is the method for using test pieces.

In the present invention, reagent components that react with alkaline creatinine to develop a color include picric acid in the Jaffe method, 3,5-dihydroxybenzoic acid in the Benedictobeu 7 method, and derivatives thereof. In addition, wetting agents, stabilizers, and the like, which will be described in detail later, may be added to the reagent components as necessary.

The alkaline component of the present invention is used for the purpose of providing a pH range suitable for the reaction between creatinine and the reagent component. A suitable pH is between 12 and 13.

As substances that can provide such a pH range, alkaline substances including various strong alkalis used in conventional liquid reaction systems such as sodium hydroxide, potassium hydroxide, and lithium hydroxide are used. can do.

The carboxylic acid derivative polymer used in combination with the alkali component in the present invention is used for the purpose of preventing alkaline denaturation of the absorbent carrier due to the above-mentioned strong alkali. is not particularly limited as long as it is alkali soluble, specifically maleic anhydride.
Isobutylene copolymer, maleic anhydride-styrene copolymer, maleic anhydride-vinyl acetate copolymer, maleic anhydride-butadiene copolymer, maleic anhydride-vinyl chloride copolymer, maleic anhydride-isoprene copolymer Union,
and maleic anhydride-vinyldene chloride copolymer.

The test piece for quantification of creatinine according to the present invention may contain a wetting agent to make the coloration uniform on the test piece, a stabilizer to stabilize the reagent components, etc., as necessary. Anionic and nonionic surfactants are particularly suitable as such wetting agents.

The test strip for quantification of creatinine according to the present invention can be prepared by utilizing not only an absorbent carrier but also a film-forming agent. That is, a film-forming agent is mixed with an alkali component, a carboxylic acid derivative polymer, and a reagent component that develops a color by reacting with creatinine, and the mixture is coated on a suitable support and then solidified.

With the test piece for quantifying creatinine according to the present invention, creatinine can be quantitatively determined by reading the degree of coloration by measuring reflectance and comparing it with a calibration curve prepared in advance. Semi-quantitative determination can also be performed by visually comparing the degree of color development with a color chart prepared in advance.

Next, a method for manufacturing a test piece according to the present invention will be described.

In the method for producing a test piece for quantifying creatinine in body fluids according to the present invention, the various components in the solvent are 5 to 20% by weight of the carboxylic acid derivative polymer, and the alkali component is 1% by weight based on the carboxylic acid residue of the carboxylic acid derivative polymer used. 0-1.
It is preferable to use about 5 times the molar amount, and 0.5 to 5% by weight of the reagent component that develops color by reacting with creatinine.

Examples of the above-mentioned absorbent carrier include filter paper, nonwoven fabric, cotton, glass fiber, etc., but filter paper is particularly preferred.An example of the method for producing the test piece for quantification of creatinine of the present invention using these absorbent carriers will be described in more detail. The following is the explanation.

That is, first, an absorbent carrier is immersed in an aqueous solution containing an alkali component and a carboxylic acid derivative, and then dried at 50 to 80°C.
Next, the obtained absorbent carrier was immersed again in an alcoholic solution of a reagent component that reacts with creatinine to develop a color, and then
It is dried at ~50°C.

The test piece thus obtained can be made into a form that is easy to handle by attaching it to a suitable support.

[Operation of the invention] The combination of an alkaline component and a carboxylic acid derivative polymer in the present invention makes it possible to utilize the alkaline component by preventing the denaturation of the absorbent carrier by the alkaline component, and is effective even when undiluted urine is used as a sample. It has the effect of providing a suitable pH range for the reaction.

On the other hand, a phenomenon has been observed in which the alkaline component adsorbs carbon dioxide in the atmosphere during storage to produce carbonic acid, resulting in a decrease in pH.

As mentioned above, since the pH range suitable for the reaction is narrow, such a phenomenon leads to deterioration of the performance of the reagent components.

However, it was unexpectedly confirmed that the carboxylic acid derivative polymer used in the present invention effectively suppresses the adsorption of carbon dioxide by alkaline components. That is, the carboxylic acid derivative polymer in the present invention contributes not only to protecting the carrier but also to improving the storage stability of the test piece itself.

Regarding the prevention of pH decrease due to carbon dioxide adsorption of alkaline components, the use of a basic component that does not contain sodium ions and an alkali-protecting polymer has been disclosed (Tokuko Sho 6l.
-6340), L The high pH value maintained by the combination of the carboxylic acid derivative polymer and the alkaline component of the present invention is due to the buffering capacity generated between the carboxylic acid residue and the alkaline component. Therefore, the presence or absence of sodium in the alkaline component is irrelevant.

The present invention will be explained in more detail with reference to Examples below.

[Example] Example 1 A chromatography filter paper (manufactured by Whatman, 3MM) was immersed in the following first impregnating liquid, and then dried at 60°C for 60 minutes, and then the obtained paper was soaked in the following second impregnating liquid. Soaked in 50
It was dried at .degree. C. for 20 minutes to obtain a test paper for quantifying creatinine containing maleic anhydride-imbutylene copolymer as a carboxylic acid derivative. After pasting this test paper on a plastic board with double-sided tape, cut it into strips with a width of 51 mm.
A test piece for quantifying creatinine according to the present invention (hereinafter referred to as test piece 1) was prepared by attaching a test paper of 5×5 intestinal layers to one end of a support made of a plastic piece having a length of 80 μm (hereinafter referred to as test piece 1).

One end 1 Impregnating liquid - 15.2 g of maleic anhydride-isobutylene copolymer and 7.5 g of sodium hydroxide are dissolved in 100 ml of distilled water.

One end 2 Impregnating liquid - Picric acid 1.0 g Sodium lauryl sulfate 0.3 g Polyvinylpyrrolidone - 300.5 g is dissolved in ethanol 1001.

Isohene (trade name) manufactured by Umbrella Talalay Isoprene Chemical ■ As a comparative example, a filter paper was immersed in the first impregnating solution in the above example except that the anhydrous marlin acid-imbutylene copolymer was removed. When an attempt was made to prepare a test paper for quantifying creatinine, the filter paper fibers were denatured by the action of alkaline components during the drying process, making it no longer usable.

Using test piece 1, a calibration curve shown in FIG. 1 was prepared using a creatinine standard solution. The measurement operation is as follows. In other words, 10JII of creatinine standard solution was dropped onto the test paper of test piece 1, and after being left at 37°C for 2 minutes, the reflectance (R-) of the coloration at 495 nm was measured using a colorimeter. /Kuberuka->junk
) is called the formula /5-(1-Roo)2/2Ro.

Convert the reflectance to /S value.

Furthermore, using actual urine as a sample, we applied the Jaffe method to automatic analysis reagent 705CRE Eiken (manufactured by Eiken Chemical ■, product name).
Taking the correlation between the value and the creatinine constant by test piece l, y = 0, 982 Y = 1.008X-11,
65 (X-automatic analysis reagent, Y-test piece 1). The measurements were carried out in the same manner as in the preparation of the calibration curve, except that the reagents for automatic analysis were used according to the specified method and the test piece l was the urine i sample.Example 2゜The composition of each impregnating solution was as follows. A test piece for quantification of creatinine (hereinafter referred to as test piece 2) according to the present invention containing maleic anhydride-imbutylene copolymer as the carvone m derivative polymer was obtained by the same operation as in Example 1, except for changing to .

-First impregnation liquid- Maleic anhydride-isobutylene copolymer - 15.2 g Sodium hydroxide 7.5 g in distilled water 1
Dissolved in 001.

One end 2 impregnation liquid - 3,5-dinitrobenzoic acid 1.5 g Tween 80 0.2 g hydroxypropyl cellulose 1.0 3 to ethanol 10
Dissolve with 01.

A calibration curve was created in the same manner as in Example 1 except that the measurement wavelength was 560 nm.
A calibration curve with linearity up to g/dl was provided. Also, regarding the correlation with automatic analysis reagents, y = 0, 958
Y=0.952X+8.5 (X-automatic analysis reagent, Y-test piece 2), showing a good correlation.

Example 3 A test piece for quantifying creatinine according to the present invention containing a maleic anhydride-styrene copolymer as the carboxylic acid derivative polymer was prepared by the same operation as in Example 1, except that the composition of each impregnating solution was changed as follows. (hereinafter referred to as test piece 3) was obtained.

1st impregnating liquid maleic anhydride 16.0 in monostyrene copolymer.

8.8g of sodium hydroxide in 1L of distilled water
Dissolve in OOml.

First and second impregnating liquid - Picric acid 1.0 g Tween 80 0.2 g Hydroxypropyl cellulose 1.0 g ethanol 100
Dissolve in 1.

SMA-Resin (trade name) manufactured by Clarei Soprene Chemical ■ A calibration curve was created in the same manner as in Example 1 except that the measurement wavelength was 490 nm.
A calibration curve with linearity up to /di was provided. Also regarding the correlation with reagents for automatic analysis.

y = 0, 972 Y-0,964X+ 17
．． 8 (X-automatic analysis reagent, Y-test piece 3).

This stability test In order to investigate the storage stability of the test piece according to the present invention, the test piece L was left under the following three conditions and changes in 1 reactivity were investigated. (200g/di) The measurement operation was carried out in the same manner as in Example 1 except that 10 pl was used. The results are shown in Table 1.

・Condition 1: Stored at 60°C for 7 days ・Condition 2: Stored at 60°C for 14 days ・Condition 3: Stored at 25°C 65% 8H for 5 hours Table 1 ・Titer: Measured value immediately after preparation was taken as 100% Time value/appearance change: Three-dimensional value of color measured by color difference meter, a
Difference in appearance color (ΔE) before and after leaving as determined from The present invention provides a test piece that enables reaction on the test piece under alkaline conditions and has excellent storage stability.

On the other hand, the concentration of creatinine in urine at any time varies depending on the degree of concentration of the urine and the degree of development of skeletal muscle, but in general, the concentration of creatinine in urine is 300 m
The calibration curve obtained with the test piece of the present invention, which is less than 0 g/dl, showed almost linearity up to 300 g/dl, and could be fully used for the determination of urinary creatinine.

As described above, according to the present invention, it is possible to provide a test piece for creatinine determination that has a high shelf life and can also be used as a sample of undiluted urine.

[Brief explanation of the drawing]

Figure 1 shows the calibration curve obtained with test piece 1, in which the vertical axis represents the /S value and the horizontal axis represents the creatinine concentration (-g
/dl).

Claims (1)

[Scope of Claims] 1) A test piece 2 for quantifying creatinine in body fluids, comprising at least a reagent component that develops color upon reaction with creatinine under alkaline conditions, an alkaline component, and an alkali-soluble carboxylic acid derivative polymer. ) The alkali-soluble carboxylic acid derivative polymer is maleic anhydride-isobutylene copolymer, maleic anhydride-isobutylene copolymer,
Styrene copolymer, maleic anhydride-vinyl acetate copolymer, maleic anhydride-butadiene copolymer, maleic anhydride-vinyl chloride copolymer, maleic anhydride-isoprene copolymer, and maleic anhydride-vinyl chloride copolymer. 3) A test piece for quantifying creatinine in body fluids according to claim 1, characterized in that the absorbent carrier is selected from the group consisting of copolymers containing at least an alkali component and an alkali-soluble carboxylic acid derivative polymer. A method for producing a test piece for quantifying creatinine in body fluids, which comprises sequentially immersing the test piece in an aqueous solvent containing the creatinine and an organic solvent containing a reagent component that reacts with at least alkaline creatinine to develop a color, and then drying the sample.