JPH01117798A - Bile acid-determining test paper - Google Patents

Abstract

PURPOSE:To obtain the title test paper which is capable of determining the bile acid in the body fluid accurately, simply in a shortened time, by allowing the carrier made of a macromolecular material to support a specific reagent composition and an additive such as ethylenediaminetetraacetic acid on the same point on the carrier. CONSTITUTION:(A) A carrier of a macromolecular material such as filter paper, nonwoven fabrics, or membrane filter of natural or synthetic fibers (such as a cellulose acetate membrane having 0.1-0.4mu pore diameter) is allowed to support, on the same point, (B) a reagent composition consisting of (a) 3alpha- hydroxysteroid dehydrogenase, 0.1-10,000 IU; (b) nicotinamide adenine dinucleotide, 0.1-100mg; (c) diaphorase, 0.1-10,000 IU and (d) tetrazolium salt, 0.1-500mg, (C) at least one selected from EDTA, ethylene glycol bis(beta- aminoethyl ether)-N,N,N',N'-tetraacetic acid or their alkali metal salts, 1-10,000mg, per 100cm<2> of the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a test strip for measuring the amount of bile acids in body fluids.

(Prior Art) Bile acids contained in bile are also contained in trace amounts in body fluids such as blood and urine. The amount of bile acids in body fluids such as kidneys changes depending on diseases of the liver system, and it is known that the amount of bile acids in blood, in particular, is a sensitive marker for such diseases. It is known that the amount of bile acids in the blood or urine increases in cases of biliary tract obstruction in infants.

Biliary obstruction occurs at a high incidence of approximately 1 in 10,000 infants, and patients require prompt surgery. Mortality rates are also high when the disease is recognized late. In order to detect such diseases early, it is desirable to accurately measure bile acids in body fluids, especially in blood, during mass medical examinations.

Methods for measuring the amount of bile acids in a sample solution containing bile acids are described, for example, in Japanese Patent Publication No. 59-13197 and Special Sale No. 1856.
-144096 and Japanese Unexamined Patent Application Publication No. 5T-151499.

According to this, first, a sample containing bile acids is subjected to heat treatment under acidic conditions (Kihan WA59-13197), or
Adding oxamic acid, pyruvic acid, etc. (sometimes open fl1
Publication No. 356-144096, JP 56-15149
No. 9) Inactivates enzymes that interfere with measurement, such as lactate dehydrogenase (LDH). This was then treated with 3α-hydroxysteroid dehydrogenase (3α-H5D) and nicotinamide adenine dinucleotide (NAD”).
, a reaction solution containing diaphorase and a tetrazolium salt is reacted under alkaline conditions at pH 8 to 9. In the presence of the hydroxyl group t13α-H5D of bile acid, it reacts with NAD+ to form a carbonyl group, producing a keto-type bile acid as follows.

NADH reacts with a tetrazolium salt, which is an electron-accepting chromogen, in the presence of diaphorase to produce formazan as follows. NADH#i is oxidized again and becomes NAD+. Redezrin may be used instead of the tetrazolium salt, in which case resorufin is produced.

NADHNAD” Tetrazolium salt The number of moles of formazan (resorufin) converted into formazan corresponds to the number of moles of NADH (that is, the number of moles of bile acid).Therefore, by measuring the absorbance of this formazan (fluorescence intensity of resorufin), It is possible to quantify bile acids.

Although bile acids in a sample can be measured with high sensitivity by such a method, it requires complicated operations because it is a measurement method that utilizes a reaction in a solution system. Therefore, it is unsuitable for mass screening or convenient detection of bile acids.

In order to solve these drawbacks, Japanese Patent Application Laid-Open No. 61-268
In Publication No. 199, 3α-H is added to a carrier made of a polymeric material.
A test strip for measuring nine bile acids, which carries a reagent composition comprising 9D%N A D'', diaphorase, and a tetrazolium salt, has been disclosed.

When this test strip comes into contact with bile acids in body fluids, the hydroxyl groups of the bile acids are converted to N in the presence of 3α-H5D, as in the solution method.
It reacts with AD+ to form a carbonyl group, producing a but-type bile acid. NADH reacts with a tetrazolium salt in the presence of diaphorase to produce formazan. NADHFi is oxidized again to NAD+. The number of moles of formazan produced is equivalent to the number of moles of FiNADH, that is, the number of moles of bile acid.

Therefore, the amount of bile acid is determined by measuring the coloration caused by formazan with the naked eye or using an optical instrument.

In this case, there are segments in the body fluid other than bile acids that can react with the reagent composition of the test strip, resulting in inaccurate measurements. Particularly in the case of body fluids derived from critically ill patients, there are many cases where the deviation from the true bile acid value is large. Components that have such an adverse effect include lactic acid and lactate dehydrogenase (LDH), alcohol and alcohol dehydrogenase, glutamate and glutamate dehydrogenase, aldehydes and aldehyde dehydrogenase, and/or formamide and formamide dehydrogenase.

For example, when lactic acid and lactate dehydrogenase (LDH) are present, they react with NAD'' in the reagent composition of the test strip,
Lactic acid produces pyruvic acid, and in the presence of the produced NADHFl and diaphorase, the tetrazolium salt is converted to formazan and the test paper is colored.

Lactic acid Pyruvate As in this example, if lactate and lactate dehydrogenase are present in the body fluid in which bile acids are to be measured, the color of the test strip will depend on the bile acids and the lactate dehydrogenase. Because the coloration caused by the dehydrogenation reaction is added, it is not possible to measure the true amount of bile acids.

In order to eliminate such drawbacks, special equipment 1@59-13197
In the publication, the sample body fluid is acidified (PHα1-6.0
) followed by heat treatment (temperature 20-45℃, time 1-30℃).
In the specification of JP-A-61-268199, it is proposed that oxamic acid and pyruvin be added to the body fluid as a sample in advance by inactivating the enzyme in the body fluid (for 1 minute).

However, such processing is complicated and troublesome for the laboratory technician, and there is a problem in that it requires extra time.

(Problems to be Solved by the Invention) The present invention solves the above conventional problems, and its purpose is to accurately and easily measure bile acids in body fluids without being affected by coexisting biological components. The purpose is to provide a method.

(Means for solving the problem) A reagent composition consisting of 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, diaphorase, and tetrazolium salt and ■ ethylenediaminetetraacetic acid (
EDTA), ethylene glycol bis(β-aminoethyl ether) N, N, N', N'tetraacetic acid (EGTA), and at least one additive selected from the group consisting of EDTA or an alkali metal salt of EGTA. The above object is achieved by a test strip for bile acid measurement, which is characterized in that it carries .

All known carriers can be used for the test strip of the present invention. That is, they are made of polymeric materials, and specifically include paper and nonwoven fabrics made of natural and synthetic fibers, as well as membrane filters. When the sample is urine or serum, paper or nonwoven fabric made of natural or synthetic fibers, such as commercially available filter paper, are preferably used. When the sample is whole blood, paper, nonwoven fabric, membrane filter, etc. made of synthetic fibers are preferably used. As a Menpreschif 4 router, the hole diameter is α1 ~ 0.4
μm cellulose acetate-based membranes are preferred. As the synthetic paper, for example, Cellbore (hydrophilic type) manufactured by Tsukisui Kagaku Kogyo (■) is suitable.

The reagent composition used in the present invention is composed of 3α-H5D and NAD, which use bile acids as substrates, diaphorase whose substrate is NADH, which is a reaction product of NAD+, and tetrazolium salt, which is a chromogenic substrate. .

Among these reagent-based compositions, NAD'', diaphorase, and chromogenic substrate not only act as 3α-1(SD(F)) to react with bile acids in body fluids to color the test strip; The test strip becomes colored when it reacts with a lactate-reducing enzyme such as lactate dehydrogenase contained in body fluids and its substrate (for example, lactic acid).

Therefore, the present inventors added ethylenediaminetetraacetic acid (EDTA) and the reagent composition to the same location on the test paper.
Ethylene glycol bis(β-aminoethyl ether)
A test paper supported with one or more additives selected from the group consisting of N, N, N', N' tetraacetic acid (EGTA), EDTA, or an alkali metal salt of #-tEGTA is prepared, and As a result, it was found that the amount of bile acids could be accurately measured without being influenced by coexisting biological components, and the present invention was completed. The amount of additive used is
The effect can be fully exhibited in the range of 1IIIf to 1o, oooq per 100sf of carrier.

When supporting additives on a carrier, EDTA and EG
When TA#iPH is 7 or less, it is difficult to dissolve in water, so adding these alkali metal salts in the form of EDTA mononatrichum, KDTA dinatrichum, EGTA dicalicum salt, etc. is easier to dissolve in water and easier to use. .

In addition, the time when the additive is supported on the carrier is not limited to which step in the manufacturing of the test strip, but the time when the additive is supported on the carrier is not limited.
It is preferable to perform this at the same time as the impregnation with an aqueous solution in which 1NAD'', diaphorase, etc. are dissolved, since the number of steps does not increase.

Next, the method for preparing and using the test strip will be explained in detail.

An aqueous solution is prepared by dissolving NAD'', 3α-H5D, diaphorase, and additives in distilled water.

After impregnating this into a carrier as described above, freeze-drying is performed. The origin of the enzyme used here is not particularly limited, but enzymes with excellent organic solvent resistance, stability over time, etc. are preferred. Among such enzymes, 3α-H5D is Pseudomonas testosterone.
diaphorase derived from Bacillus stearothermophilus.
Those derived from US) are preferably used. Nicotinamide adenine dinucleotide phos-7ate (NADP") may be used instead of NAD+. NADP+ also acts as a coenzyme like NAD+, and when reduced, it becomes reduced nicotinamide adenine dinucleotide phos-7ate (NADP"). ). 3α-H5D and diaphorase each have a concentration of 0.1 to 1000 per 100− of carrier.
01U17) percentage, NAD” (hereinafter, NAD+ is NA
DP+ and NADH may be NADPH) are supported at a ratio of α1 to 100. If the amount is too low, color development by the bile acid will not occur sufficiently, and if it is in excess, the enzymatic reaction will be inhibited by its decomposition products.

The aqueous solution may contain an activator or stabilizer for the enzyme or coenzyme. As the enzyme activator, for example, a surfactant such as Triton X-100 (trade name) is preferably used. As the enzyme stabilizer, for example, proteins such as comb serum albumin (BSA) are preferably used. When the above surfactants and proteins are added, N
These compounds include AD+ and enzymes (3α-H3D and diaphorase).

Since such surfactants and proteins do not dissolve in the non-aqueous solvent used in the step of supporting the tetrazolium salt described below, the tetrazolium salt, which is the chromogen in the non-aqueous solution, and the enzymes and coenzymes mentioned above come into direct contact. can be avoided. As a result, color development of the base during storage is suppressed.

Furthermore, a thickener may be contained. The thickener suppresses the so-called window pane phenomenon.

The window frame phenomenon is, for example, when a carrier is impregnated with the aqueous solution and dried, the solute in the aqueous solution moves to the periphery of the carrier and becomes concentrated, or when a sample solution is dropped onto the obtained test strip, NAD”, 3α-H5D contained in paper
This is a phenomenon in which reagents such as reagents migrate to the periphery of the test paper and become concentrated.

When such a window phenomenon occurs, bile acids cannot be measured accurately. Examples of the binder include methylcellulose, polyethylene glycol (PEG), and the like. When a thickener is included, the viscosity of the liquid phase impregnated into the carrier (test paper) increases, thereby suppressing the movement of solutes, and as a result, the window frame phenomenon is suppressed. The above-mentioned enzyme activators, enzyme stabilizers, thickeners, etc. are each used at a concentration of 2,001 kg per 100 cj of carrier.
ng or less, preferably at a ratio of 1A-1OOIlv.

In the freeze-drying process of a carrier impregnated with an aqueous solution containing enzymes, etc., it is important to sufficiently remove water. If water remains in the carrier, the stability of the tetrazolium salt supported in the next step will be extremely reduced.

Next, the freeze-dried carrier is impregnated with a solution of a tetrazolium salt dissolved in a non-aqueous solvent.

Examples of tetrazolium salts include nitrotetoxolicum (NTB) and nitroglutetoxolicum (NTB).
3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bis['2-(p-nitrophenyl)-5-phenyl-2H-tetrazolicumuclotide], called BT'') is preferably used.

Any non-aqueous solvent may be used as long as it can dissolve the tetrazolium salt, and alcohols such as methanol and ethanol; ethyl acetate and the like are used.

Tetrazolium salt has α1 to 5o per 100c11 of carrier.
It is carried in a proportion of oq. If the amount is too low, color development by the bile acid will not occur sufficiently, and if it is in excess, it will become difficult to dissolve in the solvent and the base color will become dark, making it difficult to distinguish the change in color tone. The carrier impregnated with the tetrazolicum salt solution is immediately dried, preferably by lyophilization.

The test paper thus obtained is cut into strips of an appropriate size and attached to the end of a stick made of plastic film to produce a test paper.

This test strip is used to measure bile acids in samples such as body fluids.

When a specimen is dropped onto a test strip, a reaction occurs according to the reaction mechanism described in the section of the prior art, producing formazan and coloring the test strip. This reaction usually occurs in 1 to 300 seconds.

Bile acid concentration in a sample is measured by the following procedure.

■ Using a suitable reflected light measuring device, the sample is dropped onto the test paper and light of a certain wavelength is irradiated to determine the intensity of the reflected light.

@ Measurement is carried out twice with different elapsed times from the drop of the sample (for example, 10 seconds and 120 seconds after the drop of the sample).

The intensity of reflected light decreases as the reaction progresses and coloration progresses.

The difference between the measured values 02 times, ie, the degree of decrease in the reflected light intensity during this period of time, is calculated as *.

■ By comparing this difference in reflected light intensity with a calibration curve that shows the relationship between the difference in reflected light intensity and bile acid concentration, which was created using a standard bile acid solution with a known concentration as a sample, Determining the acid concentration (action) When one or more additives of alkali metals are added,
Color reactions caused by substances other than bile acids can be almost completely suppressed without affecting the measurement of bile acids.

There are 0 substances other than bile acids that cause the coloration of this test strip.
Various enzymes such as the aforementioned LDH and alcohol dehydrogenase (ADH) and their substrates, reducing substances (e.g. ascorbic acid) for the 01 color substrate (tetrazolicum salt), etc. are considered. Metals are subtly involved in the reaction, as shown below.

Regarding (2), these enzymes contain divalent metal ions, and often exhibit enzyme activity in the presence of these metal ions. For example, alcohol dehydrogenase and alkaline phosphatase require zinc, amylase requires calcium, and amylase requires magnesium to express their activity. Regarding (2), for example, it is thought that the oxidizing effect of the metal is involved in the expression of the reducing effect of ascorbic acid.

On the other hand, EDTA, EGTA, or their alkali metal salts form extremely stable water-soluble complex salts with alkaline earth metals, rare earth metals, and transition metals to trap metals. In addition, by converting a small amount of metal or ion into a complex ion, there is a property that inhibits its action as a catalyst (the suppression of oxidation of ascorbic acid described below is due to this property).

Therefore, when EDTA% EGTA or their alkali metal salts are added to the test strip, the metal ion trapping action of EDTA causes dehydrogenases such as LDH% ADH, and other various substances in body fluids that affect this reagent system. Suppression of enzymatic activity of enzymes, and reducing substances such as ascorbic acid in body fluids are no longer oxidized.
It is estimated that 1J etc. will occur. Furthermore, at this time, 3α-H
Metal ion F is required for the expression of 3D and diafoduse activities.
I don't think it's related. Therefore, it is considered that the color reaction caused by substances other than bile acids could be suppressed without affecting the measurement of bile acids.

(Example) The invention will be explained below with reference to examples.

Example 1 Preparation of test strip for bile acid measurement = 3α-H3D33 I
U, diaphorase 6800IU, β-NAD"lα2
rng, EDTA-2Na (Isotope Kagaku) 50~, P
EG16tng, B5Al 00■ and Triton
10 pl of X (trade name) was dissolved in 10 liters of distilled water. This aqueous solution was filtered using 300cII filter paper (Whatmann A
3) and freeze-dried. Next, nitroprotetrazolicum (manufactured by Wako Pure Chemical Industries, Ltd., NTB) 17
) (L 034 W/W% ethanol solution IIg&
t. The freeze-dried filter paper was impregnated with this and then quickly dried. 6 test strips obtained in this way
A test paper portion was obtained by cutting into a small piece of fl×101 pieces.

These test strips were attached to the end of a stick made of 6×60+w polystyrene film with double-sided tape to obtain a test strip for bile acid measurement.

(B) Reflected light measuring device The reflected light intensity was measured using the apparatus shown in FIG.

This measuring device IFi includes a transparent plate 3 on which a test strip portion 2 for bile acid measurement impregnated with a reagent is placed, a light emitting element 4 disposed below this transparent plate 3, and a stray light prevention plate 5. It has a light receiving element 6 disposed near the light emitting element 4, and a display means 8 for numerically displaying the intensity of the reflected light from the test paper 2 detected by the light receiving element 6.

The display means BFi is electrically connected to the light-receiving element 6 via an intensifier/measuring circuit/A-D converter 7. The light emitting element 4 and the amplification/measuring circuit/A-D converter 7 are connected by a measuring switch 9. As the light emitting element 4, 500 to 600
A light emitting diode (EBG5504S manufactured by Stanley 11i) having a maximum emission spectrum near nm is used. As the light receiving element 6, a silicon photodiode (51226-5BQ manufactured by Hamamatsu Photonics Co., Ltd.), which is a photodetecting element sensitive to light having a wavelength of around 500 to 600 nm, is used. The means 8 for numerically displaying the intensity of reflected light has a built-in microprocessor.

Measuring bag r! 11, bile acids are measured as follows. A test paper section 2 for bile acid measurement is attached to the tip of a stick 21 made of polystyrene film, and a sample (urine, serum, standard solution for preparing a calibration curve, etc.) is dropped onto this test paper section 2. At the same time as the sample is dropped, a timer (not shown) is turned on, and at the same time, the test paper portion 2 is placed on the transparent plate 3 with the side facing the transparent body 3. Close the light shielding cover 22. Then, the measurement switch 9 is turned on over time to cause the light emitting element 4 to emit light. The light reflected from the test paper portion 2 is received by a light receiving element 6, passes through an amplification circuit, a measuring circuit, and an A-D converter 7, and then a display means 8 displays the reflected light intensity numerically.

(C) Creation of a calibration curve Add bile acids (sodium cholate) to normal human pool serum to determine bile acid concentrations of 0.10.25.50.100μm
A standard bile acid solution of o l// was prepared.

20 μg of the standard bile acid solution was dropped onto the test paper portion prepared in Example 1(A). 5 after 10 seconds and 120 seconds after dropping
The reflected light intensity at 40 am was measured using the reflected light measuring device of Example 1 (B), and the difference between the reflected light intensity after 10 seconds and after 120 seconds was determined from these measured values. The results are shown in the first barley.

Table 1 The calibration curve shown in FIG. 2 was obtained from the difference in reflected light intensity and bile acid concentration shown in Table 1.

D) Measurement of Bile Acid Concentration in Patient Serum Bile acid concentration in hepatitis patient serum was measured in exactly the same manner as in Example 1 (C), except that hepatitis patient serum was used instead of the standard bile acid solution.

The difference in the intensity of reflected light from 10 seconds to 120 seconds was determined by the reflected light measurement device to be 95, and by comparing this value with the calibration curve in Figure 2, the bile acid concentration was determined: Bile acid concentration #: t41 μm ol/l
! It became.

In addition, when the concentration of bile acids in this patient's serum was measured using the solution method bile acid measurement kit "Endepil" (manufactured by Dai-Kagakuyaku), it was found to be 40 μm ol/l, which was determined using the test paper of the present invention. It was in good agreement with 41 μmol/l.

Comparative Example 1 The test paper preparation method described in Example 1(A) was used.

The relationship between the standard bile acid concentration and the difference in reflected light intensity is as shown in Table 2, from which the calibration curve shown in Figure 3 was obtained.

When the bile acid concentration of the same sample as in Example 1 is measured, the difference in reflected light intensity is Vi205, and the bile acid concentration is 7 from the calibration curve.
It was found to be 8 μm ol/l.

Table 2 Example 2 In the test strip preparation method described in Example 1 (Al), E
EGTA-2N instead of DTA-2Na 50q
A calibration curve was created in the same manner as in Example 1 using a test paper prepared in exactly the same manner except that 50q1 g (1 g of Sigma) was added.Relationship between the standard bile acid concentration and the difference in reflected light intensity is as shown in Table 3.

Table 3 When using this test paper to measure the bile acid concentration in the serum of a hepatitis patient, the difference in reflected light intensity was 104, which was 4 from the calibration curve.
It was found to be 2 μm o l/l.

Furthermore, when the concentration of bile acids in this patient's serum was measured using the solution method bile acid measurement kit "Enzapile" (manufactured by Dai-Kagakuyaku), it was found to be 40 μmat/l, which was 42 μm as determined using the test paper of the present invention.

It was in good agreement with 1/l.

Comparative Example 2 When the bile acid concentration of the same sample as in Example 2 was measured using the test strip of Comparative Example 1, it was 80 μmol! /l was found.

From the above Examples and Comparative Examples, the values measured using the test strip of the present invention and the values measured using the solution method bile acid measurement kit "Enzapile" are almost equal.

On the other hand, measurements using conventional test strips that do not contain EDTA-'2Na, etc., show much larger values than those of the present invention, and in this case, the coloration due to the reaction of bile acids in the sample This indicates that the coloration is due to the reaction of coexisting components other than bile acids. That is, by adding EDTA-2N a etc. to the test paper,
It was found that the concentration of bile acids can be measured accurately.

(Effects of the Invention) According to the present invention, ■ a reagent composition consisting of 3α hydroxysteroid dehydrodanase, nicotinamide adenine dinucleotide, diaphorase, and tetrazolicum salt and ■ ethylenediaminetetraacetic acid are placed in the same place on a carrier made of a polymeric material. (EDTA), ethylene glycol bis(
β-aminoethyl ether) N, N, N', N'; and one or more additives selected from the group consisting of tetrahedral acid (EGTA), EDTA, or an alkali metal salt of EGTA. A test strip for measuring bile acids, characterized by
In this way, the amount of bile acids can be measured while excluding the influence of coexisting components in body fluids, so the amount of bile acids can be measured accurately.

Therefore, the amount of bile acids can be measured using a simple method using test strips and in a short time with an accuracy comparable to that obtained using a complicated method such as a solution method. It is highly useful for early detection of diseases during medical examinations and for emergency examinations at the bedside.

[Brief explanation of the drawing]

Fig. 1 is a schematic diagram showing the reflected light measuring device used in the present invention, and Fig. 2 shows the relationship between the standard bile acid concentration and the reflected light intensity when the standard bile acid concentration was measured using the test strip of the present invention. Figure 3 is a calibration curve showing the relationship between standard bile acid concentration and reflected light intensity when the standard bile acid concentration was measured using a conventional test strip. DESCRIPTION OF SYMBOLS 1... Bile acid measuring device, 2... Test paper part for bile acid measurement, 3... Transparent plate, 4... Light emitting element, 5... Stray light prevention plate, 6... Light receiving element, 7... Amplification/measurement circuit/A-D converter, 8... Display means, 9... Measurement switch, 21... Plastic stick, 22...
・Blackout cover. that's all

Claims (1)

[Claims] 1) (A) 3α- at the same location on a carrier made of a polymeric material
A reagent composition consisting of hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, diaphorase, and tetrazolium salt; and (B) ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(β
-aminoethyl ether) N,N,N',N'tetraacetic acid (
one or more additives selected from the group consisting of EGTA), EDTA or an alkali metal salt of EGTA,
A test strip for measuring bile acids characterized by being supported.