JPS6125498A - Reagent for quantitative determination of component in body fluid - Google Patents

Abstract

PURPOSE:To determine the minor component in a body fluid, accurately and easily, remarkably suppressing the interference reaction, by adding tartaric acid (or its alkali metal salt) to a reaction system utilizing a specific dehydrogenase in the presence of an electron transfer material and an electron acceptor. CONSTITUTION:Tartaric acid (alkali metal salt) is added to a body fluid such as blood serum, in the presence of an electron transfer material and an electron acceptor necessary for the detection and obtained by adding oxalic acid to a combination of ferric ion and a color-developing iron chelate. The amount of the tartaric acid (salt) is adjusted to give the final concentration in the reaction liquid of 0.01-1.0M. The components are made to react with each other via a dehydrogenase which necessitates free coenzyme or keeps the coenzyme as a prosthetic group. Two hydrogen atoms (or electrons) are produced per one molecule of the component by the action of the dehydrogenase. The amount of the objective component (e.g. bile acids in the serum) can be determined by determining the amount of hydrogen (or electron).

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" The present invention relates to a novel test reagent used for measuring components in biological fluids. That is, it is mainly used for clinical testing of biological fluids.

``Prior art'' Methods for measuring components in biological fluids have changed from those that require complicated extraction, concentration, and separation operations, or those that rely on chemical reactions with low specificity, to simple yet highly specific and accurate measurements. It has been a long time since the transition to enzymatic methods that made it possible. Most of these measurement methods mainly utilize one of the following two types of enzyme systems. That is, the target component is measured as the amount of hydrogen (electrons) via a dehydrogenase, and the other component is measured as the amount of hydrogen peroxide via an oxidase. The former method, which uses dehydrogenase, has been widely used since ancient times due to the abundance of available enzymes.
The measurement is performed by detecting the amount of reduction of a coenzyme coupled to an enzyme reaction or changing the electron acceptor via an electron carrier and detecting the amount of change. The present invention relates to novel reagent components for a measurement method using this dehydrogenase, electron carrier, and electron acceptor.

In this reaction mediated by an electron carrier, 92 electrons are released per component molecule by the action of dehydrogenase, and the electron acceptor is reduced via the electron carrier. Therefore, if the amount of change in the electron acceptor is monitored, a detection system will be established. This detection system can be used to electrically measure the current generated by directing the flow of electrons to an electrode system, or to cause a change in light absorption characteristics by reducing the electron acceptor, and to measure the amount of change spectrophotometrically. There are many cases. In the former case, where short-term changes are directly measured, one electrode of the battery, such as platinum or suitably treated carbon, is used as the electron acceptor, and this electrode is used as the electron acceptor, and this electrode is connected to the second electrode, such as silver/silver chloride, It is connected to platinum etc. in ferricyanide, and a salt bridge etc. connects these two electrodes. Of course, the shapes of these electrodes may vary. On the other hand, examples of electron acceptors that cause changes in the latter's light absorption properties include 2,4-dichlorophenolindophenol and the rare tetrazolium salts. It can be measured optically.

These are often accompanied by color changes in the visible region. In fact, this simple absorbance analysis is often used in the analysis of components in biological fluids in hospitals and the like. Among these, it is common to use a tetrazolium salt as an electron acceptor and use the amount of formazan produced by this reduction reaction as an indicator.

``Problems to be solved by the invention''Formazan's color reaction has the advantage of being poorly soluble, such as staining target components in tissue sections and staining electrophoresis images, but it is difficult to use in test tubes or cuvettes used for colorimetry. Because it adsorbs significantly to tubes, its use in expensive equipment such as automatic analyzers is avoided, and its usage is quite low.

Furthermore, in the case of water-soluble indicators, which exhibit a constant molar extinction coefficient, formazan is sparingly soluble, so it has the disadvantage that the molar extinction coefficient varies depending on the degree of fat solubility of the reaction solvent. (Also, the molar extinction coefficient is approximately 20,000 at most.) Therefore, it is limited to components that are contained in biological fluids at relatively high concentrations or to components to which other methods cannot be applied.

Furthermore, coenzymes, electron carriers, and
Alternatively, there may be interference reactions other than the reaction to the electron acceptor, so the measurement method is never satisfactory. Interference reactions accompanying the analysis of biological fluids are caused by dehydrogenases, electron carriers,
This is essential to a reaction system consisting of an electron acceptor, and the first object of the present invention is to provide a reagent that improves this.

Recently, a method for measuring triglycerides using F- as an electron acceptor has been proposed (Publication of Patent Publication No. 54-80192), and the application of a water-soluble chromogenic electron acceptor different from formazan to dehydrogenases has been suggested. However, in this reaction example, it is particularly easy to receive interference from components other than the target component present in the biological fluid, so even in this case, the measurable component is contained in a relatively high concentration that requires only a small amount of sample. It is limited to what can be done. Furthermore, F- itself has poor storage stability, and as a result of the formation of reddish-brown precipitates and reduction to F-, the reagent blank value increases markedly over time. As described above, it is extremely unstable as a reagent and develops color before adding the sample, so it is necessary to read the reagent blank value for each sample, making it extremely difficult to handle. These things are extremely inconvenient in daily testing. Furthermore, it is generally recognized that heavy metals containing iron have an inhibitory effect on enzymes, especially SH enzymes, causing their inactivation, and that iron chelating agents also deactivate metalloenzymes containing iron. ing. In this sense, it is difficult to apply the method using F- and an iron chelating agent to the measurement of a wide variety of components without some countermeasures.

Iron complexes as indicators of powerful hydrogen enzyme reactions have advantageous properties compared to the above-mentioned formazan. In other words, two electrons generated by the dehydrogenation reaction of one molecule of components directly reduce two molecules of Fe''+, thereby doubling the sensitivity and establishing an extremely sensitive measurement system.

Moreover, since the generated complex is water-soluble, it has the property of completely eliminating the problem of poor solubility of conventional formazan. Therefore, this coloring agent provides an advantageous method for measuring components such as the bile acids creatinine and creatine that are contained in small amounts of FF1K in biological fluids, but as mentioned above, it is susceptible to interference substances. Because it is easy to use, the reaction caused by the interfering substance often becomes larger than the reaction caused by the target component, significantly impairing the reliability of the measurement. Furthermore, this interference effect has not been fully avoided even in spectroscopic analysis methods that are already widely used as routine tests at various inspection institutions, so this interference effect, along with the numerous problems with dye systems mentioned above, must be avoided. In other words, by suppressing the interference that inherently exists in the reactions of dehydrogenases, electron carriers, and electron acceptors, we can make daily tests easier and more accurate, and also measure trace components. The practical application of dye systems that can be used for this purpose is an important theme and will make a significant contribution to practical matters.

The inventors of the present invention focused on these requirements and, as a result of intensive research, completed the present invention.

"Means for solving the problem" (and effects), namely, dehydrogenases that require free coenzymes or supplementary coenzymes in the presence of electron carriers and electron acceptors required for detection. In a method for measuring components in a biological fluid by detecting electrons generated by a reaction mediated by a dehydrogenase held as a molecular group, tartaric acid or its alkali salt is added as a reagent to suppress interference reactions other than the relevant reaction. This is a reagent for quantifying components in biological fluids, which is characterized by a kernel consisting of:

The present invention will be explained in detail below. In the present invention, when performing a series of reactions to dehydrogenase, electron carrier, and electron acceptor, if tartaric acid or its alkali salt is coexisting therein,
This makes the measurement method using dehydrogenase more accurate and reliable, since the influence of interference effects is significantly reduced. Figure 1 shows the decrease in the interference effect of 0 and 2M sodium tartrate on the serum-derived interference reaction (the graph indicated by T in the figure), in contrast to the symmetrical state (the graph indicated by C in the figure), and the absorbance (564 nm). Expressed in relation to reaction time (however, serum 100μA!
/1. (b) However, a remarkable effect is recognized.

Furthermore, in the present invention, when using F-, which is advantageous in terms of high sensitivity and water solubility as a dye system, and its color-forming chelating agent, the stability of iron itself and the relationship between F- and the iron chelating agent in the oxidized state are important. In order to maintain stability in the reduced coloring state after the stability reaction, avoid inhibition of dehydrogenase, and suppress interference reactions, it is recommended that K coexist with oxalic acid or its alkali salt for a favorable effect. can get. Figure 2 shows a situation where the presence of 1 millimolar sodium oxalate alleviates the influence of - on the change of F- to Fe+ at a final concentration of 0.2 molar and stabilizes F- (in the figure). The graph shown in FIG.

Examples of substances that are contained in biological samples and are thought to have an interfering effect include reducing substances such as ascorbic acid and bilirubin urate.

In addition, reactions of other dehydrogenases contained in the sample or aldimine adsorbing various low-molecular substances are also thought to reduce electron acceptors such as Fe''+ and cause errors.

As shown in Figure 2, Fe'' is
It gradually transforms into Fe ox, resulting in unfavorable coloring during the reaction. In addition, in weakly acidic to alkaline conditions, the hydroxo complex Fe"(OH-) is formed, and OH- crosslinks the complexes with each other, leading to polymerization and formation of precipitates. To prevent these phenomena, It is necessary to coexist with a substance that becomes a weak ligand of F- as a stabilizer,
This stabilizer affected not only the stability between F- or F- and the iron chelating agent, but also the reactivity between F- and the electron carrier, and had the effect of suppressing interference reactions. That is, the stabilizing effect of F- often tended to suppress main reactions and interference reactions. Oxalic acid was selected as the most suitable stabilizer to keep F- stable and to facilitate the flow of electrons from the electron carrier without interfering with it.

Further, to explain the present invention in detail, the final concentration in the reaction solution of tartaric acid or its alkaline salt used to suppress interference reactions in the presence of dehydrogenase, electron carrier, and electron acceptor is 0.01 to 0.01. 1.0M, solid, preferably 0.1-0.
It is 5M. The oxalic acid or its alkali salt used for the series of reactions or for stabilizing the relationship between F- and the iron chelating agent is more preferably 0.5 to 100 mM in the reaction solution.
It can be used in a concentration range of 2-50 rnM.

In spectroscopy using F- and iron chelators as electron acceptors, the iron chelators used are panphenanthroline, its sulfonic acid group, or 2-(5-bromopyridylazo)-5-(N-propyl). -N-sulfozolobylamino)aniline sodium salt (5-Br-PSAA),
3-(2-pyridyl)-5,6-bis(4-sulfophenyl)-lt 214-triazine disodium salt (
PDTS) is the most advantageous against these interference effects, and is said to proceed smoothly without affecting the dehydrogenation reaction, and is less susceptible to interference effects greater than those of the target components. This problem arises from the inherent redox potential of the complex with the chelating agent, and is not directly related to the essence of the selection of the substance and stabilizer that suppresses the interference effect of the present invention. Further, the appropriate range for carrying out the series of reactions to the dehydrogenase, electron carrier, and electron acceptor described above is in the range of 4 to 10, more preferably 6 to 9. When the above conditions are used, the stability of F- itself and the stability between F- and the iron chelating agent in the oxidized state are dramatically improved, and a series of reductive coloring reactions mediated by dehydrogenase and electron carriers occur. In this method, it is possible to accurately measure target components without being affected by interference reactions derived from biological fluids.

Trace components such as bile acids, creatine, and creatinine have traditionally been measured using the formazan color method, which has the problems mentioned above, immunological methods using antibodies, and methods using fluorescent dyes. There were difficulties in quality control regarding properties and reactivity, and special equipment such as a fluorometer was required. Compared to these prior art techniques, the present invention provides a practical solution that relies on a dehydrogenase reaction involving a wide range of target components, with the highest sensitivity and water solubility, properties that formazan lacks, using existing equipment. It provides measurement reagents.

"Example" This will be explained in more detail below using examples.

Example 1 Measurement of total bile acids in serum 1, Reagent solution A 10mM 3-(2-pyridyl)-5,6-bis(4-sulfophenyl)-11214-)riazine disodium salt (PDTS) % 0.42M mouth, shell salt and 80
0.01 containing μM methoxyphenazine methosulfate.
1M Triethanolamine Buffer -7.0 Solution B 0, 55mM Iron, Uban, 37mM Sodium Oxalate, 1% Trlton
．． 0.1 M containing 4 UARl, and 6 NAD
) Lichnolamine buffer pH 7.0 Solution C Reagent solution D similar to solution B except for 3α-hydroxysteroid dehydrogenase I'rIM diethylenetriaminepentaacetic acid (,I)TP
A) 0.1M 3.3-& Methylglutarate buffer pH 6,02 containing A) Serum Zooμ11 solution A 0.25m/ml was added to a test tube and Zooμ11 solution B O, 25rRl was added to the test tube for exactly 10 minutes at 37°C. Immediately after the reaction, 1 ml of solution D is added and the temperature is returned to room temperature. At the same time, the absorbance at 564 nm was measured using a sample blank that was operated in the same manner by replacing solution B with solution CK. Next, the total bile acid concentration in the serum sample is calculated by comparing the absorbance shown by a known sodium cholate solution treated in the same manner as the serum sample and the absorbance obtained by subtracting the sample blank absorbance from the measured absorbance. Under the above conditions, the overall absorbance response is <in> as shown in Figure 3.
Levels of up to 00 μM total bile acids were observed.

Example 2 Measurement of creatine in serum 1, Reagent solution A 10mM 3-(2-pyridyl)-5+ 6-bis(4-
Sulfophenyl)-1,2,4-) riazine disodium salt (PDTS), 0.42Me! , shell salt and 80
μM Methoxyphena Zone - Contains Methosulfate 0
．． 1 M) rittanolamine buffer p) 17.0 Solution B Z2mM iron, upan, 90mM oxalic acid, 1% Trl
ton X-1001 li/4'-ze 400 U, 4/,
Creatinase 70 Ufil, Delcosine Dehydrogenase 30 U/kl.

0.IM) containing 0.1% (W/V) bovine albumin)
Rittanolamine buffer pJ (7,0 Solution C 30mM sodium phosphate buffer pH 7,02 Procedure method Serum 20μ11 solution A
After adding O.5m and reacting at 37°C for 10 minutes, immediately add solution 01- and return to room temperature. Separately, the same procedure was performed without adding serum, and the reagent blank was used as 564.
Measure the absorbance in nm. Next, the creatine content of the serum sample is calculated by comparing the measured absorbance with the absorbance shown by a solution of known creatine content treated in the same manner as the serum sample.

Under the above conditions, as shown in FIG. 4, a direct f9 absorbance response was observed up to the level of creatine of 20.4t.

"Effects of the Invention" As is clear from the above, according to the present invention, in the presence of an electron carrier and an electron acceptor required for detection, a dehydrogenase that requires a free coenzyme or a coenzyme as a prosthetic molecule In a method for measuring components in a biological fluid by detecting electrons generated by a reaction mediated by a dehydrogenase that is retained as a group,
By using tartaric acid or its alkali salt, interfering reactions other than the relevant reaction can be significantly suppressed, making measurement accurate and simpler.

[Brief explanation of drawings]

Figure 1 is a graph showing the inhibitory effect of sodium tartrate on serum-derived interference reactions, and Figure 2 is a graph showing the influence of sodium oxalate on the stability of F-. FIG. 3 is a graph showing a calibration curve when measuring bile acids, and FIG. 4 is a graph showing a calibration curve when measuring creatine. Figure 1: Time taken (all) Figure 3: Sales (A M) Figure 4: OIQ 20 Rishi Aaman (m9/d)

Claims (2)

[Claims] (1) In the presence of an electron carrier and an electron acceptor required for detection,
In a method for measuring components in a biological fluid by detecting electrons generated by a reaction mediated by a dehydrogenase that requires a free coenzyme or a dehydrogenase that retains a coenzyme as a prosthetic group, the reaction 1. A reagent for quantifying components in biological fluids, which comprises adding tartaric acid or an alkali salt thereof as a reagent for suppressing other interference reactions. (2) The electron acceptor required for detection is ferric ion (Fe^
2) and a color-forming iron chelating agent, and further comprises adding oxalic acid as a stabilizing reagent to the reagent for quantifying components in biological fluids.