JPH0683679B2 - 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 and Actions for Solving Problems) Reagent consisting of a 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, diaphorase and tetrazolium salt in the same place on a carrier made of a polymer material System composition and ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) N, N, N ', N'tetraacetic acid (EGTA), EDTA
Or one or more additives I selected from the group consisting of alkali metal salts of EGTA and haxamic acid, pyruvic acid, oxalic acid, 8-chloro-4hydroxy-5-methylquinoline-3carboxylic acid and salts thereof. The above object is achieved by a test paper for measuring bile acid, which is loaded with one or more additives II selected from the group consisting of:

More preferably, a reagent system composition comprising i 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, diaphorase and tetrazolium salt and polyethylene glycol diacetate at the same location on the carrier made of a polymeric material. (EDTA), ethylene glycol bis (β-aminoethyl ether) N, N, N ', N'tetraacetic acid (EGTA), one or more additives selected from the group consisting of EDTA or alkali metal salts of EGTA I and at least one additive II selected from the group consisting of haxamic acid, pyruvic acid, oxalic acid, 8-chloro-4hydroxy-5-methylquinoline-3carboxylic acid and salts thereof. A and the ones supported on part A from 3α-hydroxysteroid dehydrogena Component lacking Zenomi the above object is achieved by the bile acid measuring test paper characterized by comprising a combination of the supported portion B.

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.

In the above reagent system composition, nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) instead of NAD ⁺
May be used. Like NAD ⁺ , NADP ⁺ also acts as a coenzyme, and when reduced, produces reduced nicotinamide adenine dinucleotide phosphate (NADPH), and NADPH also acts as a substrate for diaphorase. In the following explanation,
NAD ⁺ may be NADP ⁺ and NADH may be NADPH.

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,
One or more additives I selected from the group consisting of N ', N'tetraacetic acid (EGTA), EDTA or alkali metal salts of EGTA I
And oxamic acid, pyruvic acid, oxalic acid, 8-chloro-4
One or more additives selected from the group consisting of hydroxy-5-methylquinoline-3carboxylic acid and salts thereof
When a test paper carrying II and II was prepared and bile acid in the body fluid was measured, it was found that the amount of bile acid could be accurately measured without being affected by coexisting biological components, and the present invention was completed.

The amount of additive used is such that Additive I is 1 mg to ¹ per 100 cm ² of the carrier.
The effect of Additive II can be sufficiently exerted within the range of 0.1 to 500 mg per 100 cm ² of the carrier.

Upon loading the additive I on the carrier, EDTA and EGTA
When the pH is 7 or less, it is difficult to dissolve in water, so it is easier to use these alkali metal salts such as EDTA monosodium, EDTA disodium, EDTA dipotassium salt, etc. .

In addition, when the additive is loaded on the carrier, (a) a method that is performed at the same time as impregnating a part of the reagent composition, and (b) a method that is performed at the same time when impregnating all of the reagent composition. There is a method of impregnating only the solution of the additive separately from the time of impregnation, and a method of impregnating the additive I and the additive II simultaneously or separately is conceivable, but either method can be used. is there. However, a method in which the additive I and the additive II are simultaneously dissolved in an aqueous solution in which a reagent-based composition containing no tetrazolium salt is dissolved to impregnate them simultaneously is preferable 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, additive I and additive II 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. 3α-HSD and diaphorase are each 0.1 to 10000 IU per 100 cm ² of the carrier,
NAD ⁺ is loaded at a rate of 0.1 to 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.

According to the present invention, when the additive I and the additive II were added to the reagent-based composition of the conventional bile acid measurement test paper, there was no effect on the measurement of bile acid, and coloration was caused by other than bile acid. The reaction could be suppressed almost completely.
This effect will be considered next.

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 contained in the additive I 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 additive I was able to suppress the color reaction due to substances other than bile acid without affecting the measurement of bile acid.

In addition, additives II oxamic acid, pyruvic acid, oxalic acid, 8
All of -chloro-4hydroxy-5 methylquinoline-3carboxylic acid and salts thereof have an LDH inhibitory action. Therefore, when these are used, the reaction of lactic acid in the body fluid using LDH as a catalyst is suppressed.

As described above, the effects of Additive I and Additive II more completely suppress the color reaction due to substances other than bile acid, as compared with the case where each is used alone.

In the present invention, in order to always accurately and accurately quantify bile acid in any sample, it is preferable to perform a blank measurement which is usually performed in analysis of general biochemical substances.
That is, a control test paper (for blank measurement) was prepared, and one sample was used as a test paper for the bile acid test paper as described above and a test paper for control (blank measurement) as the test paper. Is a measurement method corresponding to the amount of.

The following test paper is used for such a measuring method.

At the same location on the carrier made of a polymeric material, 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, a reagent system composition consisting of diaphorase and tetrazolium salt and ethylenediaminetetraacetic acid (EDTA), Ethylene glycol bis (β-aminoethyl ether) N, N, N ′, N ′ tetraacetic acid (EGTA), EDTA
Or one or more additives I selected from the group consisting of alkali metal salts of EGTA and haxamic acid, pyruvic acid, oxalic acid, 8-chloro-4hydroxy-5-methylquinoline-3carboxylic acid and salts thereof. Part A in which one or more additives II selected from the group consisting of
And a part B carrying a component lacking only 3α-hydroxysteroid dehydrogenase from the part carried on part A, and a test paper for measuring bile acid.

The test paper is prepared as follows.

The part A of the test paper is prepared by supporting the reagent composition A, the additive I and the additive II on the carrier as described above. Part B of the test paper is prepared by similarly supporting a component lacking only 3α-HSD from the component supported on part A on a carrier. The test paper portions A and B thus obtained are cut into strips of appropriate size and attached to the ends of a stick made of a plastic film to produce a test paper. At this time, the portions A and B of the test paper may be attached to separate sticks, or may be attached to both ends of one stick.

Using this test paper, 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) This measurement is performed on the same specimen for the test paper parts A and B, and the degree of decrease in reflected light intensity is measured by Ref.
(A) and Ref (B).

E = Ref (A) -Ref (B), Ref (A) -R prepared using a standard bile acid solution of known concentration as a sample
The bile acid concentration in the sample is determined by comparison with a calibration curve showing the relationship between ef (B) and the bile acid concentration.

When this test strip is used, the part A of the test strip is thus
The amount of bile acid in the sample can be determined by using the value obtained by subtracting the degree of coloration generated on the part B of the test paper from the coloration generated on the test paper. Impact is removed.

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

Example 1 Preparation of test strip for bile acid measurement: 3α-HSD30IU, diaphorase 6800IU, β-NAD ⁺ 10.2 mg, EDTA-2Na (manufactured by Dojindo) 1
00mg, sodium oxamate 5mg, PEG16mg, BSA100mg
And 10 μm of TritonX (trade name) were dissolved in 10 ml of distilled water. A 300 cm ² filter paper (Whatmann No. 3) was impregnated with this aqueous solution and freeze-dried. Next, a 0.034 W / W% ethanol solution of nitroblue tetrazolium (NTB manufactured by Wako Pure Chemical Industries, Ltd.) was prepared and impregnated into the above-mentioned freeze-dried filter paper and then quickly dried. The test paper thus obtained was cut into 6 mm × 10 mm pieces to obtain a portion A of the test paper.

Next, a part B of the test paper was prepared in the same manner except that 3α-HSD was removed from the above reagent composition.

The portions A and B of these test papers were attached to the ends of 6 × 60 mm polystyrene film sticks with double-sided tape to obtain test papers 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 /.

The standard bile acid solution (20 μm) was dropped on the portions A and B of the test paper prepared in Example 1 (A). 10 seconds after dropping and 120 seconds after 555
The reflected light intensity in nm was measured using the reflected light measuring device of Example 1 (B), and the difference between the reflected light intensities after 10 seconds and 120 seconds was determined from the measured values. Let Ref (A) and Ref (B) be the difference between parts A and B of the test paper, and Δ = Ref
When (A) -Ref (B) was obtained, it was as 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. If only Ref (A) is taken as the reflected light intensity difference, it corresponds to a test paper without blank measurement,
Taking Ref (A) -Ref (B) corresponds to the case of performing blank measurement.

(D) Measurement of Bile Acid Concentration in Patient Serum Bile acid concentration in serum of liver disease patient was carried out in exactly the same manner as in Example 1 (C) except that serum of patient with liver disease was used as the sample instead of standard bile acid solution. It was measured.

As serum of patients with liver disease, two samples were measured. The respective specimen names are designated as specimen 1 and specimen 2, and the difference in reflected light intensity is shown in Table 2.

By comparing the values of Ref (A) and ΔRef (A) -Ref (B) with the calibration curve of FIG. 2, the bile acid concentration was obtained as follows.

Measured only from Ref (A) Specimen 1 = 29μmol / Specimen 2 = 58μmol / Measured from Ref (A) -Ref (B) Specimen 1 = 27μmol / Specimen 2 = 50μmol / In addition, the concentration of bile acid in this patient's serum When using the solution method bile acid measurement kit "Enzabairu" (manufactured by Daiichi Pure Chemicals), sample 1 = 28 µmol /, sample 2 = 51 µmol /
Became.

Comparing this value with the value obtained with the test paper of the present invention, for the sample 1, the measured value only from Ref (A) (29 μmol /
) And Ref (A) -Ref (B) measurements (27 μmo
It matched well with l /). For sample 2, Ref (A)
Good agreement with the measured value from Ref (B) (50 μmol /), but deviated considerably from the measured value from Ref (A) (58 μmol /).

Comparative Example 1 In the method for preparing the test paper described in Example 1 (A), ED
Example 1 was carried out using test papers prepared in exactly the same manner except that TA-2Na and sodium oxamate were removed.
The bile acid concentration of the same sample as in Example 1 was measured in the same manner as in.

The relationship between the standard bile acid concentration and the difference in reflected light intensity is shown in Table 3, 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 as shown in Table 4.

By comparing the values of Ref (A) and Δ = Ref (A) -Ref (B) with the calibration curve in FIG. 3, the bile acid concentration was obtained as follows.

Measured only from Ref (A) Sample 1 = 55 μmol / Sample 2 = 110 μmol / Measured from Ref (A) -Ref (B) Sample 1 = 32 μmol / Sample 2 = 56 μmol / Compared with, when both the samples 1 and 2 were measured only from Ref (A), they were significantly different from the measured values of Enzabile.
Even when measured from f (A) -Ref (B), it was considerably different from the measured value of Enzabile.

As described above, from Example 1 and Comparative Example 1, when the test paper added with EDTA-2Na and sodium oxamate was used, Re
When measuring only from f (A), depending on the sample (eg,
Specimen 2) There are some deviations from the values measured with enzabyle, but the values are much closer to the values measured with enzabyle than the values measured with test papers that do not contain these additives.

In addition, when measured from Ref (A) -Ref (B), EDTA-2N
It was found that in the case of the test papers to which a and sodium oxamate were added, a measurement value closer to the measurement value of enzabyle was obtained compared to the test papers to which these were not added.

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 except that 100 mg of EGTA-2Na (manufactured by Sigma) was added instead of 100 mg of TA-2Na.

Using this test paper, the bile acid concentration of the same sample as in Example 1 was determined to be as follows.

Measured only from Ref (A) Sample 1 = 30 μmol / Sample 2 = 60 μmol / Measured from Ref (A) -Ref (B) Sample 1 = 29 μmol / Sample 2 = 51 μmol / Example 3 Described in Example 1 (A). A calibration curve was prepared in the same manner as in Example 1 except that 5 mg of pyruvic acid was added instead of 5 mg of sodium oxamate in the method for preparing the test paper.

Using this test paper, the bile acid concentration of the same sample as in Example 1 was determined to be as follows.

Measured only from Ref (A) Sample 1 = 27 μmol / Sample 2 = 57 μmol / Measured from Ref (A) -Ref (B) Sample 1 = 28 μmol / Sample 2 = 49 μmol / Example 4 Described in Example 1 (A). In the test paper preparation method, ED
Instead of TA-2Na 100 mg, EGTA-2Na 100 mg (manufactured by Sigma), using test paper prepared in exactly the same manner except that pyruvic acid 5 mg was added instead of sodium oxamate 5 mg. A calibration curve was prepared in the same manner as in 1.

Using this test paper, the bile acid concentration of the same sample as in Example 1 was determined to be as follows.

Measured only from Ref (A) Sample 1 = 29 μmol / Sample 2 = 61 μmol / Measured from Ref (A) -Ref (B) Sample 1 = 29 μmol / Sample 2 = 52 μmol / Above, any of Examples 2, 3 and 4 The measured values of the test paper also showed almost the same results as in Example 1 using EDTA-2Na and sodium oxamate as additives.

(Effects of the Invention) The test strip for measuring bile acid of the present invention is a carrier made of a polymer material, wherein 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, diaphorase and tetrazolium are present at the same position. Reagent-based composition consisting of salt and ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) N,
One or more additives I selected from the group consisting of N, N ', N'tetraacetic acid (EGTA), EDTA or alkali metal salts of EGTA, and haxamic acid, pyruvic acid, oxalic acid, 8-chloro-4 Since at least one additive II selected from the group consisting of hydroxy-5-methylquinoline-3carboxylic acid and salts thereof is carried, it is characterized in that it is a coexisting component in body fluids. Excluding the influence, the amount of bile acid can be measured, and the amount of bile acid can be accurately measured.

Further, using the control test paper of the present invention, Ref (A) -Ref
If the bile acid concentration is obtained from (B), the effects of various coexisting substances in the body fluid can be further suppressed, and the amount of bile acid in specimens of all kinds of hepatobiliary system diseases can be always accurately measured. Simultaneous reproducibility is also improved.

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 standard bile acid concentration and a difference in reflected light intensity when a standard bile acid solution is measured with the test paper of the present invention. A calibration curve showing the relationship, and FIG. 3 is a calibration curve showing the relationship between the standard bile acid concentration and the reflected light intensity difference 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 (2)

[Claims] 1. A carrier made of a polymer material is placed at the same position on the carrier.
Reagent system composition consisting of α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, diaphorase and tetrazolium salt and ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) N , N, N ′, N ′ Tetraacetic acid (E
GTA), one or more additives I selected from the group consisting of alkali metal salts of EDTA or EGTA and haxamic acid, pyruvic acid, oxalic acid, 8-chloro-4hydroxy-5
A test strip for measuring bile acid, which is loaded with one or more additives II selected from the group consisting of methylquinoline-3carboxylic acid and salts thereof. 2. A carrier made of a polymer material is placed at the same position on the carrier.
Reagent system composition consisting of α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, diaphorase and tetrazolium salt and ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis (β-aminoethyl ether) N , N, N ′, N ′ Tetraacetic acid (E
GTA), one or more additives I selected from the group consisting of alkali metal salts of EDTA or EGTA and haxamic acid, pyruvic acid, oxalic acid, 8-chloro-4hydroxy-5
Part A carrying one or more additives II selected from the group consisting of methylquinoline-3carboxylic acid and salts thereof, and lacking only 3α-hydroxysteroid dehydrogenase from those carried on part A A test paper for measuring bile acid, characterized by comprising a combination with a component B carrying a component.