JPH0683678B2 - Bile acid test strip - Google Patents

Description

TECHNICAL FIELD The present invention relates to a test strip for measuring the amount of bile acid in body fluid.

(Prior Art) Bile acids contained in bile are contained in trace amounts in body fluids such as blood and urine. It is known that the amount of bile acid in such body fluid varies depending on hepatobiliary disease, and that the amount of bile acid in blood is a sensitive marker for such a disease. For example, it is known that in infants with biliary obstruction, the amount of bile acid in blood or urine increases. Biliary obstruction occurs at a high rate of 1 in 10,000 infants and patients require immediate surgery. Mortality rates are higher if the recognition of the disease is delayed. In order to detect such diseases at an early stage, it is desired to accurately measure bile acids in body fluids, particularly blood, at the time of mass screening.

A method for measuring the amount of bile acid in a sample solution containing bile acid is disclosed, for example, in Japanese Examined Patent Publication No. 59-13197, Japanese Unexamined Patent Publication No. 56-144096, and Japanese Unexamined Patent Publication No. 56-151499. According to it, first, a sample containing bile acid is heat-treated under acidic conditions (Japanese Patent Publication No. 59-13197), or oxamic acid, pyruvic acid, etc. are added (Japanese Patent Publication No. 56-144096, Japanese Patent Publication No. (Kaisho 56-151499) Lactate dehydrogenase (LDH)
Inactivate enzymes that interfere with the measurement, such as. Then, the 3α-hydroxysteroid dehydrogenase (3
α-HSD), nicotinamide adenine dinucleotide (N
A reaction solution containing AD ⁺ ), diaphorase and a tetrazolium salt is reacted under alkaline conditions of pH 8-9. The hydroxyl group of bile acid reacts with NAD ^{+ in} the presence of 3α-HSD to become a carbonyl group, and produces a keto-type bile acid as follows.

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

The number of moles of formazan (resorufin) produced corresponds to the number of moles of NADH (that is, the number of moles of bile acids). Therefore, it is possible to quantify bile acid by measuring the absorbance of this formazan (fluorescence intensity of resorufin).

Although bile acid in a sample can be measured with high sensitivity by such a method, it requires a complicated operation because it is a measurement method utilizing a reaction in a solution system. Therefore, it is unsuitable for mass screening and simple detection of bile acids.

In order to solve such drawbacks, Japanese Patent Laid-Open No. 61-268199 discloses that a carrier made of a polymer material has 3α-HSD, NAD ⁺ ,
A test strip for measuring bile acid, which carries a reagent composition comprising diaphorase and a tetrazolium salt, has been disclosed. When this test paper comes into contact with bile acid in body fluid, the hydroxyl group of bile acid reacts with NAD ^{+ in} the presence of 3α-HSD to form a carbonyl group to form a keto-type bile acid, as in the case of the solution method. NA
DH reacts with a tetrazolium salt in the presence of diaphorase to form formazan. NADH is oxidized again to NAD ⁺ . The number of moles of formazan produced corresponds to the number of moles of NADH, that is, the number of moles of bile acids. Therefore, the coloration due to this formazan is measured with the naked eye or an optical instrument to measure the amount of bile acid.

In this case, there is a component in the body fluid other than bile acid that can react with the reagent-based composition of the test strip, and as a result, the measurement may be inaccurate. Especially in the case of body fluids derived from critically ill patients, there are many cases in which the deviation from the true bile acid value becomes large. Examples of such components that have adverse effects include lactic acid and lactate dehydrogenase (LDH), alcohol and alcohol dehydrogenase, glutamic acid and glutamate dehydrogenase, aldehydes and aldehyde dehydrogenase, and / or formamide and formamide dehydrogenase.

For example, if lactic acid and lactate dehydrogenase (LDH) are present,
Lactic acid produces pyruvic acid by reacting with NAD ⁺ in the reagent-based composition of the test strip, and the produced NADH causes the tetrazolium salt to formazane in the presence of diaphorase to color the test strip.

When lactic acid and lactate dehydrogenase are present in the body fluid for which bile acid is to be measured, as in this example, the color of the test strip is decolorized by lactate dehydrogenase with lactic acid dehydrogenated by bile acid. The true amount of bile acid cannot be measured because the color is added due to the reaction.

In order to eliminate such a defect, in JP-B-59-13197, by making a body fluid as a sample acidic (PH 0.1 to 6.0) and then heat treating (temperature 20 to 45 ° C., time 1 to 30 minutes), Inactivating the enzyme in the body fluid in advance,
In the specification of -268199, it is proposed to add oxamidic acid, pyruvic acid and the like to a body fluid as a sample to inhibit the LDH reaction and the like.

However, such processing is complicated and troublesome for the inspection engineer, and there is a problem that extra time is required.

(Problems to be Solved by the Invention) The present invention is to solve the above-mentioned conventional problems, and an object thereof is to accurately and easily measure bile acids in body fluids without being affected by coexisting biological components. To provide a method.

(Means for Solving Problems) A reagent system composition comprising 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, diaphorase and tetrazolium salt and ethylenediaminetetraacetic acid (EDTA) at the same location on a carrier made of a polymer material, Ethylene glycol bis (β-aminoethyl ether) N, N, N ', N'tetraacetic acid (EGTA), EDTA or E
The above object is achieved by a test paper for measuring bile acid, which is loaded with one or more additives selected from the group consisting of alkali metal salts of GTA.

As the carrier used in the test paper of the present invention, all known carriers can be used. That is, they are made of a polymer material, specifically, papermaking or non-woven fabric made of natural or synthetic fibers, as well as a membrane filter. When the sample is urine or serum, paper making or non-woven fabric made of natural or synthetic fibers such as commercially available filter paper is preferably used. When the sample is whole blood, papermaking or non-woven fabric made of synthetic fiber, membrane filter and the like are preferably used. As a membrane filter, the hole diameter is 0.1-0.4μ
m cellulose acetate based membranes are preferred. As synthetic paper, for example, Selpore (hydrophilic type) manufactured by Sekisui Chemical Co., Ltd. is suitable.

Reagent composition, as used in the present invention, 3.alpha.-HSD and NAD ⁺ to a bile acid as a substrate, a further reaction product of NAD ⁺ N
It is composed of diaphorase with ADH as a substrate and tetrazolium salt as a chromogenic substrate.

Of these reagent-based compositions, NAD ⁺ , diaphorase and chromogenic substrate not only react with bile acids in body fluids by the action of 3α-HSD to cause the test paper to color, but also lactic acid dehydration contained in the body fluids. The test paper is colored by reacting with a redox enzyme such as an elementary enzyme and its substrate (eg, lactic acid).

Therefore, the inventors of the present invention, together with the reagent composition at the same location of the test paper, ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) N, N,
Test paper carrying one or more additives selected from the group consisting of N ', N'tetraacetic acid (EGTA), EDTA or alkali metal salts of EGTA was prepared, and bile acids in body fluids were measured. However, it was found that the amount of bile acid can be accurately measured without being affected by the coexisting biological components, and the present invention was completed. The amount of additives used is ¹ mg to 10,00 per 100 cm ² of the carrier.
The effect can be fully exhibited in the range of 0 mg.

When loading the additives on the carrier, EDTA and EGTA
When it is 7 or less, it is difficult to dissolve in water, so it is easier to dissolve in water and easier to use if it is added in the form of these alkali metal salts such as EDTA monosodium, EDTA disodium, and EDTA dipotassium salt.

Further, the timing of loading the additive on the carrier is not limited to which step of the test paper production, but 3α-HSD, N
It is preferable to perform it at the same time as impregnating the aqueous solution in which AD ⁺ , diaphorase, etc. are dissolved, because the number of steps does not increase.

Next, the method for preparing and using the test paper will be described in detail.

An aqueous solution is prepared by dissolving NAD ⁺ , 3α-HSD, diaphorase and additives in distilled water. This is impregnated into the carrier as described above, and then freeze-dried. The origin of the enzyme used here is not particularly limited, but an enzyme excellent in organic solvent resistance and stability over time is preferable. Preferable examples of such an enzyme include those derived from Pseudomonas testosteroni as 3α-HSD, and those derived from Bacillus stearothermophilus as diaphorase. Nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) may be used instead of NAD ⁺ . Like NAD ⁺ , NADP ⁺ also acts as a coenzyme, and when reduced, produces reduced nicotinamide adenine dinucleotide phosphate (NADPH). 3α-HSD and diaphorase are NAD ⁺ (hereinafter, NAD ⁺ may be NADP ⁺ , respectively) at a ratio of 0.1 to 10,000 IU per 100 cm ² of the carrier.
H may be NADPH) is loaded at a rate of 0.1-100 mg. When the amount is too small, the color development by bile acid does not occur sufficiently, and when the amount is excessive, the decomposition product inhibits the enzymatic reaction.

The above aqueous solution may contain an activator or stabilizer of an enzyme or a coenzyme. As the enzyme activator, a surfactant such as Triton X-100 (trade name) is preferably used. A protein such as bovine serum albumin (BSA) is preferably used as the enzyme stabilizer. When the above surfactants and proteins are added, NAD ⁺
Enzymes (3α-HSD and diaphorase) are included in these compounds.

Such surfactants and proteins do not dissolve in the non-aqueous solvent used in the step of supporting the tetrazolium salt described below, so the tetrazolium salt, which is a chromogen in a non-aqueous solution, directly contacts the enzyme or coenzyme. You can avoid doing it. As a result, the coloration of the base during storage is suppressed.

Further, a thickener may be contained. The thickener suppresses the so-called window frame phenomenon. The window frame phenomenon is, for example, when the carrier is impregnated with the aqueous solution and dried, the solute in the aqueous solution moves to the periphery of the carrier and is concentrated,
This is a phenomenon in which a reagent such as NAD ⁺ , 3α-HSD contained in the test paper migrates to the peripheral area of the test paper and is concentrated when the sample solution is dropped on the obtained test paper. When such a window frame phenomenon occurs, the measurement of bile acid cannot be performed accurately. Examples of the thickener include methyl cellulose and polyethylene glycol (PEG). When the thickener is contained, the viscosity of the liquid phase impregnated in the carrier (test paper) increases, so that the movement of the solute is suppressed, and as a result, the window frame phenomenon is suppressed. The enzyme activator, enzyme stabilizer, thickener, etc. are each 200 mg or less per 100 cm ^{2 of the} carrier, preferably 1 to 100
It is carried in the proportion of mg.

Thus, in the freeze-drying process of the carrier impregnated with the aqueous solution containing the enzyme and the like, it is important to sufficiently remove water. If water remains on the carrier, the stability of the tetrazolium salt supported in the next step will be extremely reduced.

Next, the lyophilized carrier is impregnated with a solution of a tetrazolium salt dissolved in a non-aqueous solvent. The tetrazolium salt is called nitrotetrazolium blue (NTB) or nitroblue tetrazolium (NBT) 3.
3 '-(3,3'-dimethoxy-4,4'-biphenylene) -bis [2- (p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride] is preferably used.

The non-aqueous solvent may be any solvent that can dissolve a tetrazolium salt, and alcohols such as methanol and ethanol; ethyl acetate and the like are used.

The tetrazolium salt is supported at a rate of 0.1 to 500 mg per 100 cm ² of the carrier. If the amount is too small, the color development by bile acid does not occur sufficiently, and if the amount is too large, it becomes difficult to dissolve in the solvent, and the color of the base becomes darker, which makes it difficult to distinguish the discoloration of the color tone.
The carrier impregnated with the tetrazolium salt solution is dried quickly, preferably by freeze-drying.

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

This test paper is used to measure bile acid in a sample such as body fluid.

When the test sample is dropped on the test paper, the reaction by the reaction mechanism described in the section of the prior art occurs, formazan is generated, and the test paper is colored. This reaction usually takes 1 to 300 seconds.

The bile acid concentration in the sample is measured by the following procedure.

A) Using a suitable reflected light measuring device, irradiate the test paper portion onto which the specimen has been dropped with light of a certain wavelength and obtain the reflected light intensity.

(2) This measurement is performed twice while changing the elapsed time from dropping the sample (for example, 10 seconds and 120 seconds after dropping the sample).

The reflected light intensity decreases as the reaction progresses and the color develops.

(C) The difference between the two measured values, that is, the degree of decrease in the reflected light intensity within this elapsed time is obtained.

D) By comparing this difference in reflected light intensity with a standard curve prepared in advance using a standard bile acid solution of known concentration as a sample and showing the relationship between the difference in reflected light intensity and bile acid concentration, the bile in the sample was compared. Determine the acid concentration.

(Effect) According to the present invention, when EDTA, EGTA or one or more additives of these alkali metals are added to the reagent composition of the conventional test strip for measuring bile acid, there is no effect on the measurement of bile acid. It was possible to almost completely suppress the reaction of color development other than bile acid without the addition of bile acid.

Other than bile acids, the causative substances that cause the coloration of this test strip are (a) various enzymes such as LDH and alcohol dehydrogenase (ADH) and their substrates, and (b) reducing substances for chromogenic substrates (tetrazolium salts) (eg ascorbine). Acid), and so on.

The metal is delicately involved in the reaction of these color-causing substances, as shown below. That is, as for a, these enzymes contain divalent metal ions, and in many cases, the enzyme activity is expressed in the presence of these metal ions. For example, alcohol dehydrogenase and alkaline phosphatase require zinc, amylase requires calcium, and enolase requires magnesium for its activity expression. Regarding b, for example, it is considered that the oxidation action of metal is involved in the expression of the reducing action of ascorbic acid.

On the other hand, EDTA, EGTA or an alkali metal salt thereof forms an extremely stable water-soluble complex salt with an alkaline earth metal, a rare earth or a transition metal and captures the metal. Further, by making a trace amount of a metal or ion into a complex ion, it has a property of hindering its action as a catalyst (oxidation of ascorbic acid shown below is due to this property).

Therefore, when EDTA, EGTA or an alkali metal salt thereof is added to the test strip, the metal ion-trapping action of EDTA or the like causes dehydrogenases such as LDH and ADH and other various substances in the body fluid that affect this reagent system. It is presumed that the inhibition of the enzymatic activity of the enzymes and the inhibition of the reducing reaction of the chromogenic substrate by ascorbic acid, etc., due to the fact that the reducing substance such as ascorbic acid in the body fluid is not oxidized.
At this time, it seems that metal ions are not involved in the activity expression of 3α-HSD and diaphorase. Therefore, it is considered that the color reaction due to substances other than bile acid could be suppressed without affecting the measurement of bile acid.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 (A) Preparation of test strip for bile acid measurement: 3α-HSD33IU, diaphorase 6800IU, β-NAD ⁺ 10.2 mg, EDTA-2Na (manufactured by Dojindo) 50 mg, PEG 16 mg, BSA 100 mg and Triton X (trade name) 10 μ It was dissolved in 10 ml of distilled water. 300c of this aqueous solution
An m ² filter paper (Whatmann No. 3) was impregnated and freeze-dried. Next, nitroblue tetrazolium (Wako Pure Chemical Industries,
A 0.034 W / W% ethanol solution of (NTB) was prepared, and this freeze-dried filter paper was impregnated with the solution and then immediately dried. The test paper thus obtained was cut into small pieces of 6 mm × 10 mm to obtain a test paper portion. Replace these test strips with 6
A stick of double-sided 60 mm polystyrene film was attached to the end of the stick with double-sided tape to obtain a test paper for measuring bile acid.

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

This measuring device 1 includes a transparent plate 3 on which a test paper portion 2 for measuring bile acid impregnated with a reagent is placed, a light emitting element 4 arranged below the transparent plate 3, and a stray light prevention plate 5. It has a light receiving element 6 arranged in the vicinity of 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 8 is electrically connected to the light receiving element 6 through the amplification / measurement circuit / A / D converter 7. Light-emitting element 4 and amplification
The measurement circuit and A / D converter 7 are connected by a measurement switch 9. As the light emitting element 4, a light emitting diode (EBG5504S manufactured by Stanley) having a maximum emission spectrum in the vicinity of 500 to 600 nm is used. As the light receiving element 6, a photo detecting element having sensitivity to light having a wavelength of about 500 to 600 nm, a silicon photodiode (S1 manufactured by Hamamatsu Photonics KK
226-5BQ) is used. The means 8 for numerically displaying the intensity of the reflected light has a built-in microprocessor.

Bile acid is measured using the measuring apparatus 1 as follows. At the tip of the stick 21 made of polystyrene film,
Stick the test paper part 2 for bile acid measurement, and
A sample (urine, serum, standard solution for creating a calibration curve, etc.) is added dropwise to. At the same time when the sample is dropped, a timer (not shown) is turned on, and at the same time, the test paper portion 2 side is placed on the transparent plate 3 so as to face the transparent body 3. Close the light shield cover 22. Then, the measuring switch 9 is turned on with time, and the light emitting element 4 is caused to emit light. The light reflected by the test paper portion 2 is received by the light receiving element 6, and the reflected light intensity is numerically displayed by the display means 8 through the amplification / measurement circuit / A / D converter 7.

(C) Preparation of calibration curve Bile acid (sodium cholate) was added to normal human pool serum to prepare a standard bile acid solution having a bile acid concentration of 0, 10, 25, 50, 100 μmol /.

20 μl of the standard bile acid solution was dropped on the test paper portion prepared in Example 1 (A). The reflected light intensity at 555 nm after 10 seconds and 120 seconds from the dropping 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 measured from the measured values. I asked. The results are shown in Table 1.

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

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

The difference in reflected light intensity from 10 seconds to 120 seconds from the reflected light measurement device is 95
The bile acid concentration was calculated by comparing this value with the calibration curve in FIG. 2 and the bile acid concentration was 41 μmol /.

In addition, the concentration of bile acid in the serum of this patient was determined to be 40 μmol / when measured using a solution method bile acid measurement kit “Enzabayle” (manufactured by Daiichi Pure Chemicals), and 41 μmol / determined with the test paper of the present invention Well matched

Comparative Example 1 In the method for preparing the test paper described in Example 1 (A), ED
Bile acid concentrations of the same samples as in Example 1 were measured in the same manner as in Example I using test papers prepared in exactly the same manner except that TA-2Na was omitted.

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

When the bile acid concentration of the same sample as in Example 1 was measured, the difference in reflected light intensity was 205, and from the calibration curve, the bile acid concentration was 78 μmol.
/ Was asked.

Example 2 In the method for preparing the test strip described in Example 1 (A), ED
A calibration curve was prepared in the same manner as in Example 1 using a test paper prepared in exactly the same manner, except that 50 mg of EGTA-2Na (manufactured by Sigma) was added instead of 50 mg of TA-2Na. The relationship between the standard bile acid concentration and the reflected light intensity difference is shown in Table 3.

When the bile acid concentration of hepatitis patient serum was measured using this test paper, the difference in reflected light intensity was 104, which was 42 μm from the calibration curve.
I got ol /.

Further, the concentration of bile acid in the serum of this patient was determined to be 40 μmol / when measured using a solution method bile acid measurement kit “Enzabaile” (manufactured by Daiichi Pure Chemicals), and 42 μmol / was determined using the test paper of the present invention. Well matched

Comparative Example 2 Using the test paper of Comparative Example 1, the bile acid concentration of the same sample as in Example 2 was measured and found to be 80 μmol /.

From the above Examples and Comparative Examples, the value measured by the test paper of the present invention and the value measured by the solution method bile acid measuring kit "Enzabairu" are almost equal.

On the other hand, what was measured with a conventional test paper containing no EDTA-2Na, etc., shows a much larger measured value than that of the present invention, in this case, due to the coloration due to the reaction of bile acid, in the sample This indicates that coloration is added due to the reaction of coexisting components other than bile acid. That is, EDTA-2N is added to the test paper.
It was found that the concentration of bile acid can be accurately measured by adding a or the like.

(Effects of the Invention) According to the present invention, a reagent system composition comprising 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, diaphorase and tetrazolium salt and ethylenediaminetetraacetic acid (EDTA) at the same location on a carrier made of a polymer material, Ethylene glycol bis (β-
Aminoethyl ether) N, N, N ', N'tetraacetic acid (EGTA), E
One or more additives selected from the group consisting of alkali metal salts of DTA or EGTA and a test paper for measuring bile acid, which is characterized by being carried, and thus, coexisting components in body fluids Since the amount of bile acid can be measured excluding the effect of, the amount of bile acid can be accurately measured.

Therefore, the amount of bile acid can be measured with the accuracy equivalent to the value obtained by the complicated method such as the solution method in a short time in a simple method such as test paper, and the method of the present invention It is highly useful for early detection of illness in medical examinations and for bedside emergency tests.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a reflected light measuring device used in the present invention, and FIG. 2 is a relationship between standard bile acid concentration and reflected light intensity when a standard bile acid solution is measured with the test paper of the present invention. FIG. 3 is a calibration curve showing the relationship between the standard bile acid concentration and the reflected light intensity when the standard bile acid solution was measured with the test paper of the conventional method. 1 ... Bile acid measuring device, 2 ... Bile acid measuring test paper part, 3 ... Transparent plate, 4 ... Light emitting element, 5 ... Stray light prevention plate, 6 ... Light receiving element, 7 ... Widening Measuring circuit / A / D converter, 8 ... Display means, 9 ... Measuring switch, 21 ...
Plastic stick, 22 …… Shading cover.

Claims (1)

[Claims] 1. A carrier made of a polymeric material is provided at the same position on the carrier.
A reagent system composition comprising α-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 (EGTA), E
A test paper for measuring bile acid, which is loaded with one or more additives selected from the group consisting of alkali metal salts of DTA or EGTA.