JPH01117799A - Test paper for measuring living body components - Google Patents

Abstract

PURPOSE:To obtain the title test paper capable of determining desired living body components in body fluid accurately and simply in a shortened time, by allowing a carrier of a macromolecular material to support, on its same point, a reagent composition which develops colors by the reaction with the subject components among the living body components and a specific coloring inhibitor. CONSTITUTION:(A) A carrier made of a macromolecular material such as filter, nonwoven fabrics, membrane filter (for example, a cellulose acetate membrane having 0.1-0.4mu pore diameter) is allowed to support, on the same point, (B) a reagent composition which develops colors by reactions with the subject components among the living body components, and is composed of (a) 0.1-10,000 IU of 3alpha-hydroxysteroid dehydrogenase, (b) 0.1-100mg of nicotinamide adenine dinucleotide, (c) 0.1-10,000 IU of diaphorase, and (d) 0.1-500mg of tetrazolium salt and (C) 0.1-500mg of a substance which inhibits the main components among the components participating the color reactions other than the objective components such as an inhibitor of lactic dehydrogenase (for example, pyruvic acid), based on 100cm<2> of the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a test strip capable of measuring the amount of biological components, and particularly to a test strip for measuring bile acids.

(Prior art) In clinical biochemical tests, the amount of biological components such as glucose and protein in urine or blood (hereinafter collectively referred to as body fluids) is determined based on the degree of coloration due to reaction with the biological components. Test strips are widely used to measure

A general embodiment of the method of using this test strip will be described. The test strip is made up of a carrier made of a polymeric material supporting a reagent-based composition that reacts with the target component to be measured to develop a color.

In the measurement, the test paper is brought into contact with body fluid, and the component to be measured therein reacts with the reagent composition of the test paper, causing coloration.

Next, the amount of the component to be measured is determined by measuring the degree of coloration with the naked eye or using an optical instrument and comparing it with a coloration sample or calibration curve prepared in advance.

Conventionally, when measuring target components in body fluids using such test strips, coloration occurs due to the reagent composition of the test strip reacting with components other than the target components to be measured.
There is a drawback that the amount of the component to be measured may not be accurately measured.

For example, this will be explained in the case of measuring the amount of bile acids, which are markers of hepatobiliary diseases.

The test strip has a reagent composition consisting of 3α-hydroxysteroid dehydrodanase (3α-H3D), nicotinamide adenine dinucleotide (NA D+), shea water 2-ase, and tetrazolium salt supported on the surface of the carrier. When this comes into contact with bile acids in body fluids, the hydroxyl group of the bile acid reacts with NAD+ in the presence of 3α-H5D to become a carbonyl group, producing the following keto-type bile acid.

N A D HFi') reacts with a tetrazolium salt in the presence of aphorase to yield formazan as follows.

NADH is oxidized again to NAD+.

Tetrazolium Salt Formazan The number of moles of formazan produced corresponds to the number of moles of NAD)I (the number of moles of bile acid).

Therefore, the amount of bile acid is determined by measuring the coloration caused by this 9-formaden with the naked eye or using an optical instrument. in this case,
There are components other than bile acids in body fluids 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 adverse effects 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. be.

For example, when lactic acid and lactate dehydrogenase (LDH) are present, the lactic acid reacts with NAD+ in the reagent composition of the test strip to generate pyruvate, and in the presence of the generated NADHH and diaphorase, the tetrazolium salt is converted to formazan. The test paper is then colored.

Lactic acid Tetrazolium pyruvate salt Formazan As in this example, when lactic acid and lactate dehydrogenase are present in the body fluid in which bile acids are to be measured, the coloring of the test strip depends on the bile acids. Due to the addition of color due to the reaction of dehydrogenase, it is not possible to measure the true amount of bile acids. PHα1-aO) and then heat treatment (temperature 20-45°C, time 1-30 minutes) to inactivate enzymes in body fluids in advance, as described in the specification of JP-A-61-268199. It has been proposed that oxamidic acid, pyruvic acid, etc. be added to the body fluid sample in advance to inhibit the LD)I reaction.

However, such detailed processing is complicated and troublesome for the laboratory technician, and also requires additional 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 components to be measured in biological components without being affected by coexisting biological components. The goal is to provide test strips that can be used for measurements.

(Means for solving the problem) In a test paper that measures the amount of a biological component based on the degree of coloration due to reaction with the biological component, a test strip that reacts with the target component of the regenerated biological component and develops color at the same location. a reagent-based composition, and (
B) The above object is achieved by a test strip for measuring biological components, which is characterized by carrying a substance that inhibits the color reaction of the main component among the components involved in the color reaction other than the target component. Ru.

Jikai 1@61-268199 attempts to solve the problem by adding a substance that inhibits this reaction to the sample in order to avoid coloration due to the reaction of components other than the target component to be measured. However, as mentioned above,
Adding a substance that inhibits the reaction to the sample one by one is
It is cumbersome and takes extra time. In the present invention, a substance that inhibits reactions other than the target component to be measured is added in advance to the reagent-carrying portion of a test strip for measuring biological components, and the above purpose is achieved without any processing of the sample. It is something to do. For example, if there is a main component B%C%D of the components involved in the color reaction other than the measurement target component A in the sample, the test paper should be prepared in advance. Only A can be accurately measured using a test strip for measuring biological components in which a substance that inhibits the reaction of B, C%D, etc. is added.

All known carriers can be used for the test strip of the present invention. That is, they are made of polymeric materials, specifically 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 membrane filter, the hole diameter is α1 to A4.
μm cellulose acetate-based membranes are preferred. As the synthetic paper, for example, Serbia (hydrophilic type) manufactured by Tsukisui Kagaku Kogyo H is suitable.

The reagent composition used in the test strip of the present invention varies depending on the object to be measured, but for example, it includes a redox reaction system consisting of an enzyme and a coenzyme whose substrate is the component to be measured in the specimen, and the coenzyme. It consists of a redox reaction system consisting of an enzyme and a chromogenic substrate that use enzyme reaction products as substrates.

A case in which bile acids in body fluids are measured will be described below as a representative example of the present invention.

The reagent-based composition used is a 3α-
It is composed of diaphorase whose substrate is NADH, which is a reaction product of H5D and NAD+, and tetrazolium salt, which is a chromogenic substrate.

Among these reagent-based compositions, NAD'', diaphorase, and chromogenic substrate not only react with bile acids in body fluids to color the test strip through the action of 3α-H5Df), but also react with lactic acid dehydration contained in body fluids. It reacts with redox enzymes such as hydrogen enzymes and their substrates (such as lactic acid), causing the test strip to change color.

When bile acids in body fluids are measured using test strips for measuring bile acids, the amount of bile acids may be larger than that obtained by chromatography or immunochemical measurement. It is often seen in severe liver diseases, and sometimes the value can be 2 to 3 times higher.

The reason for this is thought to be that LDH and lactic acid in the body fluid react with the test paper, resulting in an apparently large value.

In this way, when measuring bile acids, "the main components of the components involved in the color reaction other than the components to be measured" include, for example, lactic acid and LDH.

These include alcohol and alcohol dehydrogenase.

Therefore, the present inventors added an LDH inhibitor to the test paper and found that this phenomenon could be avoided.

LDH inhibitors include pyruvic acid, oxamidic acid, oxalic acid, and 8-chloro-4hydroxy-5methylquinoline-3.
Examples include carboxylic acids or their salts.

For bile acid measurements, the amount of inhibitor added is 10 Gc of carrier.
per d (11 to 5001 ng).

The inhibitor is supported on the carrier at the same time as impregnating a part of the reagent-based composition that reacts with the target component to be measured, or at the same time as the entire reagent-based composition is impregnated. Method, θ Any method can be used, such as impregnating only the inhibitor solution separately from the time of impregnating the reagent composition, but method ① or @ is preferable because it requires fewer steps. For example, in the method (■), 3α-H5D is added to a carrier made of a polymeric material in the process of manufacturing test strips.
, NAD'', diaphorase, etc., may be added to the aqueous solution in which the membrane is impregnated.

Next, a method for preparing the test strip of the present invention and an example of its use will be explained in detail regarding a test strip for bile acid measurement.

An aqueous solution is prepared by dissolving NAD+, 3α-H3D, diaphorase and LDH inhibitor in distilled water. After impregnating this into a carrier, 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. Such an enzyme, 3α-H3D, is Pseudomonas testosteroni (Pseudomonas testosteroni).
oni), and the diaphorase derived from Bacillus stearothermophilus.
Stearothermophilus) derivatives are preferably used. Nicotinamide adanine dinucleotide phosphate (NADp) instead of NAD+
+) may be used. NADP+ also acts as a coenzyme like "NAD", and when reduced, it produces reduced FM, -cotinamide adenine dinucleotide phosphate (NA
DPH). 3α-H5D and diaphorase at 1-100 OOIU each per 100aII of carrier.
NAD+ (hereinafter, NAD+ may be NADP+, and NADH may be NADPH) at a ratio of
It'1 (LX ~ 100q) is supported. If it is too little, color development by bile acid will not occur sufficiently, and if it is too much, the enzymatic reaction will be inhibited by its decomposition products.

Additives may be contained in the aqueous solution. Examples of additives include activators and stabilizers for enzymes and coenzymes. As an enzyme activator, for example, Triton X-100
Surfactants such as (trade name) are preferably used. As an enzyme stabilizer, for example, comb serum albumin (
Proteins such as BSA) are preferably used. When the above surfactants and proteins are added, NAD+ and enzymes (3
α-H3D and diaphorase) are included among these compounds.

Since such surfactants and proteins do not dissolve in the non-aqueous solvent used in the step of supporting tetrazolicum salt, which will be described later, the enzymes and coenzymes mentioned above come into direct contact with the tetrazolicum salt, which is a chromogen, in the non-aqueous solution. can be avoided. As a result, the color development of the base material is suppressed during storage.
Furthermore, a thickener may be contained as an additive.

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, This is a phenomenon in which reagents such as NAD+ and 3α-H9D contained in the paper 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 thickener include methylcellulose and polyethylene glycol (PEG). 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. Additives such as enzyme activators, enzyme stabilizers, thickeners, etc. are each
001 FIIJ or less, preferably 1 to 100 μm.

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.

Tetrazolium salts include 3,3'-(:I@3'-dimethoxy-4,4'-biphenylene)-bis called nitrotetrazolicum (NTB) or nido, loprotetrazolicum (NBT). (2-(p-nitrophenyl)-5-7enyl-2H-ftzolicum chloride) 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 is a carrier of 100 - 1~500t
It is supported at a ratio of ng. 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, irradiate light of a certain wavelength onto the part of the test paper onto which the specimen has been dropped, and determine the intensity of the reflected light.

(2) Perform this measurement twice, changing the elapsed time from dropping the sample (for example, 10 seconds and 120 seconds after dropping the sample).

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

The difference between the measured values 02 times, that is, the degree of decrease in the reflected light intensity within this elapsed time is determined.

■ 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, Determine acid concentration.

The above explanation describes the use of the test strip of the present invention to measure the concentration of bile acids in body fluids as a representative example, but it can also be widely applied to the measurement of other biological components using the same measurement principle. It is self-evident that this is the case (action). <This is a biological component measurement test strip that is loaded with a substance that inhibits the color reaction of the main component among the components involved. Target components can be measured accurately without the influence of coexisting substances.

For example, if you use a test strip for measuring bile acids, a test strip loaded with a reagent composition that reacts with bile acids to develop color, and an LDH inhibitor, the test strip for measuring bile acids and body fluids will react. When the LDH inhibitor supported on one test paper is LDH
%NAD+ etc. and sterile complex (abortive co
mplex) and L (Example) The present invention will be described below with examples.

Example 1 Preparation of test strip for measuring bile acid = 3α-H8D33IU
, diaphorase 68001U, β-NAD"1Q211
9, pyruvate 5rII9, PEGI6■, B5A10
0V and 10 μt of TritonX (trade name) were dissolved in 10 μm of distilled water. A 300 csf filter paper (Whatmann A 3 ) was impregnated with this aqueous solution and freeze-dried. Next, a 4% w/w ethanol solution of nitroprotetrazolicum (manufactured by Wako Pure Chemical Industries, Ltd., NTB) was prepared, and the freeze-dried filter paper was impregnated with this, and then quickly dried. The test paper obtained by heating it in this way is
It was cut into small pieces of m×10+w to obtain test paper portions. These test strips were attached to the end of a 6×60 gm polystyrene film stick using double-sided tape to obtain a test strip for bile acid measurement.

CB+ Reflected light measuring device The reflected light intensity was measured using the apparatus shown in FIG.

This measuring device 1 includes a transparent plate 3 on which a test strip portion 2 for bile acid measurement impregnated with a reagent is placed, and a lower part S of this transparent plate 3.
The intensity of the reflected light from the test paper 2 detected by the light-emitting element 4 arranged in ζ, the light-receiving element 6 arranged near the light-emitting element 4 via the stray light prevention [5], and the light-receiving element 6 is numerically calculated. and display means 8 for displaying the information.

The display means 8 is electrically connected to the light receiving element 6 via an amplification/measuring circuit/AD converter 7. Light emitting element 4 and width increaser
The measurement circuit/A-D converter 7 is connected through a measurement switch 9. As the light emitting element 4, SOO~600n
A light emitting diode (EBG5504S manufactured by Stanley Corporation) having an IC emission spectrum maximum near m is used. The light-receiving element 6 is an optical detector element having optical sensitivity at wavelengths around 500 to 600 nm. A recon photodiode (51226-5BQ manufactured by Hamamatsu Photonics) is used. The means 8Iζ for numerically displaying the intensity of the reflected light has a built-in microprocessor.

Bile acids are measured using the measuring device 1 as follows. At the tip of the stick 21 made of polystyrene film,
Paste the test strip part 2 for bile acid measurement, and
#c Drop the solution (urine, In1 test, standard wave for creating a calibration curve, etc.). An unillustrated tie occurred at the same time as the sample was dropped! - is turned on and simultaneously placed on the transparent plate 3 with the test paper portion 2 side facing the transparent body 3V. Close the light shielding cover 22. Then, turn on the measurement switch 9 over time.
The light emitting element 4 is caused to emit light. The light reflected by the test paper portion 2tc is received by the light receiving element 6, passes through an amplification/measuring circuit/A-D conversion 117, and is caused to numerically display the reflected light intensity on the display means 8.

(Creation of C1 calibration curve Normal human pool blood 111E bile acid (sodium cholate)
and bile acid concentration 0, 10, 25.501100
A standard bile acid solution of pm, OL'L was prepared.

20 tons of standard bile acid solution was dropped onto the test paper portion prepared in Example 1 (8). 5 after 10 seconds and 120 seconds after dropping
The reflected light intensity at 40 nm was measured using the reflected light measuring device of Example 1 (11), and the difference between the reflected light intensity at 10 seconds and 12 oe* was determined from these measured values.

-Table 1- and the results are shown.

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

@Measurement of Bile Acid Concentration in Serum of a Patient The concentration of bile acid in the serum of a hepatitis patient was measured in exactly the same manner as in Example 1 (C), except that the hepatitis patient's own sample was used instead of two standard bile acid solutions.

The difference in reflected light intensity from 10 seconds to 120 seconds was determined by the reflected light measurement device to be 117, and by comparing this value with the calibration curve in Figure 2, the bile acid concentration was determined to be 45 μmol/l. .

In addition, when the concentration of bile acids in this patient's serum was measured using the solution method bile acid measurement kit "Endepile" (Dai-ichi Chemicals Department), it was found to be 47 μmoSit, and 45 μrnoL' determined using the test strip of the present invention. There was good agreement with L.

Comparative Example 1 The same specimen as in Example 1 was prepared in the same manner as in Example 1 using a test paper prepared in exactly the same manner as described in Example ICA) except that pyruvic acid was omitted. The relationship between the standard bile acid concentration and the difference in reflection intensity is shown in Table 2, and from this the calibration curve shown in Figure 3 was obtained.

When measuring the bile acid concentration of the same sample as in Example 1, the difference in reflected light intensity was 225, and the bile acid concentration was 89 from the calibration curve.
μmoνL was found.

Table 2 Example 2 A test prepared in exactly the same manner as described in Example 1 except that 1ζ and 5 μ of sodium oxinamate were added in place of pyruvic acid 5119. A calibration curve was created in the same manner as in Example 1 using paper.

Table 3 shows the relationship between the standard bile acid concentration and the difference in reflected light intensity.

(Leaving space below) Table 3 Using this test paper to measure the bile acid concentration in the serum of a hepatitis patient, the difference in 1 reflected light intensity was 120, which was 5% from the calibration curve.
It was found to be 2 μmot/l.

In addition, when the concentration of bile acids in this patient's serum was measured using a solution method bile acid measurement kit [Enzapile J (II-Chemicals Department), it was found to be 54 μm0vt, which was 52-moνt determined using the test strip of the present invention. It was a good match.

Comparative Example 2 When the bile acid concentration of the same sample as in Example 2 was measured using the test paper of Comparative Example 1, it was found to be 98 μmoL/Z.

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, those measured using conventional test strips that do not contain LDH inhibitors show much higher values than those of the present invention, and in this case, the coloring due to the reaction of bile acids 1
This shows that coloration is added due to the reaction 1ζ between lactic acid and LDH.

That is, by adding an inhibitor of ζLDH to the test paper, it was possible to accurately measure the concentration of bile acids.

(Effects of the Invention) According to the present invention, a test strip for measuring the amount of a biological component based on the degree of coloration due to reaction with the biological component: (4) Purpose of measurement in the biological component. A reagent-based composition that reacts with the components to form a color, and (B) a substance that inhibits the color reaction of the main component among the components that participate in the color reaction other than the target component to be measured are supported. Since the test strip for measuring biocomponents is characterized by the following, it is possible to measure the amount of the target biocomponent while excluding the influence of coexisting components in body fluids.
Therefore, it is possible to accurately measure the amount of the target biological component using a simple method using a test strip, and in a short time, with an accuracy comparable to that obtained using a complicated method such as a solution method. The method of the present invention has high utility value in the early detection of diseases in mass medical examinations and in emergencies at the bedside.

In particular, when used to measure bile acids in body fluids, it is useful for early detection of hepatobiliary diseases.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram showing the reflected light measuring device used in the present invention. Figure 2 shows the relationship between standard bile acid concentration and reflected light intensity when a standard bile acid solution is 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 a standard bile acid solution is 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/measuring circuit/A-D converter, 8... Display means, 9... Measurement switch, 21... Pukstic stick, 22...
・Blackout cover. that's all

Claims (1)

[Claims] 1) In a test paper for measuring the amount of a biological component based on the degree of coloration due to reaction with the biological component, (A) coloration due to reaction with the target component of the biological component at the same location; (B) a substance that inhibits the color reaction of the main component among the components involved in the color reaction other than the component to be measured;
A test strip for measuring biological components, characterized in that it is supported. 2) The biological component according to claim 1, wherein the substance that inhibits the color reaction of the main component among the components involved in the color reaction other than the component to be measured is a reaction inhibitor of lactate dehydrogenase. Test paper for measurement. 3) The test strip for measuring biological components according to claim 2, wherein the lactate dehydrogenase reaction inhibitor is pyruvic acid, oxamidic acid, oxalic acid, or a salt thereof. 4) The reagent composition that reacts with the target component to be measured in the biological component to develop a color is composed of 3α-hydroxysteroid dehydrogenase, nicotinamide adenine dinucleotide, diaphorase, and tetrazolium salt, and it does not react with the target component to be measured. The test paper for measuring biological components according to claim 1, wherein the substance that inhibits the color reaction of the main component among the components involved in the color reaction is pyruvic acid, oxamic acid, oxalic acid, or a salt thereof.