JPS63245696A - Method for measuring ceruloplasmin activity - Google Patents

Abstract

PURPOSE:To rapidly and accurately measure ceruloplasmin activity useful for clinical tests of anemia, nephrosis syndrome, etc., by adding complex ions of bivalent iron into a specific reaction system. CONSTITUTION:A reaction system reagent selected from the group of 4- aminoantipyrine/aniline derivative, 4-aminoantipyrine/phenol derivative and benzothiazolinone hydrazone derivative/aniline derivative systems is prepared so as to provide conditions of pH4-6 of the reagent, 0.02-0.2M concentration of a buffer solution, such as KH2PO4, 1-20mM concentration of the 4- aminoantipyrine and 0.1-2mM concentration of benzothiazolinone hydrazone derivative. K4Fe(CN)6, etc., is then added so as to contain >=1muM complex ions of bivalent iron in this reagent to afford a reagent for measurement. The resultant reagent is subsequently mixed and reacted with a ceruloplasmin-containing test solution and absorbance of the reaction solution is measured to calculate the ceruloplasmin activity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for accurately and rapidly measuring ceruloplasmin activity.

[Prior Art] Ceruloplasmin present in mammalian blood is a copper protein contained in the α-globulin fraction electrophoretically. The copper protein mentioned above is generally said to have a close relationship with iron metabolism, and therefore, by measuring the activity of ceruloplasmin, diseases related to iron metabolism such as Wilson's disease, anemia, nephrotic syndrome, gallbladder disease, and pregnancy can be detected. Obtaining information useful for diagnosis is of extremely high clinical significance.

In principle, there are two methods for measuring ceruloplasmin.
Traditionally, there are two methods: immunological methods and chemical methods.
Streets are being carried out.

Among these methods, the immunoassay method is complicated and requires a long time for analysis, and the amount of protein detected is independent of whether it is active or inactive, and it is difficult to measure ceruloplasmin activity. This gave inaccurate results.

On the other hand, as a typical example of chemical measurement, Robin (R
ovin) colorimetric method. This method uses p-phenylenediamine to stain and measure oxidase activity. This method also requires more than 30 minutes for analysis, and it is also necessary to obtain measurement results for a sample blank separately, which is time-consuming. Furthermore, the reagent used in this method (generally p-phenylenediamine dissolved in lI acetic acid solution) is not only unstable and must be prepared for each analysis, but also the p-phenylenediamine has safety and health concerns. is also considered to be an extremely inconvenient substance. Therefore, this method cannot be considered sufficient as a routine testing method either.

To improve the drawbacks of Robin's colorimetric method,
For example, a technique as shown in Japanese Patent Laid-Open No. 57-68797 has already been proposed. This method measures ceruloplasmin using 4-aminoantipyrine (or 3-methyl-2-benzothiazolinone hydrazone) and an N-substituted aniline compound as substrates, and by using these substances, p - It eliminates the drawbacks caused by phenylenediamine.

However, this method had a decisive drawback in that it was inferior in sensitivity compared to the case of using p-phenylenediamine.

[Problems to be Solved by the Invention] The present invention has been made to solve the above-mentioned drawbacks of the prior art, and its purpose is to provide a method that can quickly and accurately measure the activity of ceruloplasmin. Our goal is to provide the following.

[Means for Solving the Problems] In order to achieve the above object, the inventors of the present invention have made various considerations, and as a result of further intensive research, if appropriate reaction conditions are set,
We have discovered that ceruloplasmin activity can be quantitatively analyzed quickly and accurately, and have finally completed the method of the present invention.

That is, the method of the present invention uses any system selected from the group consisting of 4-aminoantipyrine/aniline derivative system, 4-aminoantipyrine/phenol derivative system, and benzothiazolinone hydrazone derivative/aniline derivative system to produce ceruloplasmin. When measuring activity, pH: 4.0
~6.01 Concentration of buffer: 0.02M ~0.2M Concentration of 4-aminoantipyrine: 1-20 a+M.

Concentration of benzothiazolinone hydrazone derivative = 0.1~
This is a method for measuring ceruloplasmin activity, the gist of which is to measure ceruloplasmin in a reaction system that has been adjusted to satisfy each condition of 2mM and contains 1 μM or more of divalent iron complex ions.

[Action] Specific examples of the aniline derivatives used in the present invention include:
N-Methyl-N-hydroxymethyl-3-methylaniline, N-ethyl-N-hydroxyethyl-3-methylaniline, N-ethyl-N-hydroxyethyl-3-ditylaniline, N-methyl-N- and droxyethyl -3-
Methylaniline, N-methyl-N-hydroxypropyl-3-methylaniline, N-ethyl-N-hydroxypropyl-3-methylaniline, N-methyl-N-hydroxyethyl-3-ditylaniline, N-propyl-N −
Hydroxyethyl-3-methylaniline, N-methyl-
N-hydroxyethyl-3-propylaniline, N,N
-bis(β-hydroxyethyl)-3-methylaniline, N,N-bis(β-hydroxypropyl)-3-methylaniline, N,N-dimethyl-3-methylaniline, N,N-dimethyl-3- Dithylaniline, N,N-dimethyl-3-pro and luaniline, N,N-diethyl-3
- methylaniline, "N, N-diethyl-3-ditylaniline, N, N-dipropyl-3-methylaniline,"
N-ethyl-N-(β-methanesulfonamidoethyl)
-m-Toluidine, N-ethyl-N-(β-acetamidoethyl)-3-methylaniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diisopropylaniline, N-methyl-N- Hydroxyethylaniline, N-ethyl-N-hydroxyethylaniline,
N-ethyl-N-sulfopropyl-m-)-luidine,
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, N-ethyl-N-(2-bitroxy-3-sulfopropyl)-m-anisidine, N-ethyl-N-(2 -hydroxy-3-sulfopropyl)aniline, 3,5-dimethoxy-N-ethyl-N-(2-
hydroxy-3-sulfopropyl)aniline, 3.5
-dimethyl-N-ethyl-N-(2-hydroxy-3-
sulfopropyl)aniline, N-ethyl-N-sulfopropyl-m-anisidine, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, etc. can be mentioned.

Specific examples of phenol derivatives include p-chlorophenol, p-bromophenol, 3.5-dichlorophenylsulfonic acid, and 3-hydroxy-2,4,6-triiodobenzoic acid.

Furthermore, a specific example of the penzothialinone hydrazone derivative includes 3-methyl-2-benzothiazolinone hydrazone.

There are no particular restrictions on the concentrations of the aniline derivatives and phenol derivatives used, but approximately 0.1 to 20 mM is optimal.

When using 4-aminoantipyrine, its concentration is 1
~20mM is required. This has a concentration of 1m
If it is less than M, the required sensitivity will not be achieved, while if it exceeds 20mM, the reagent blank will become large and will be impractical. Further, when using a benzothiazolinone hydrazone derivative, it is necessary to adjust its concentration to 0.1 to 2 mM. This is because if the concentration is less than 0.1 mM, the required sensitivity will not be achieved, while if it exceeds 2 mM, the reagent blank will become large and will be impractical.

The pH of the reagent used in the method of the present invention is adjusted using an i buffer, and the pH value must be in the range of 4.0 to 6.0. This is the maximum sensitivity and optimum p of the enzyme.
H. This is because it is optimal for color stability. The buffer to be used is not limited in any way as long as it can set the pH of the reagent to 4.0 to 6.0.
Phosphoric acid, acetic acid.

tartaric acid, citric acid, succinic acid, dimethyl geltaric acid,
Buffers such as phthalic acid and glycine are suitable. The concentration of these buffer solutions needs to be 0.02M to 0.2M. This is because if the concentration of the buffer solution is less than 0.02M, it cannot function as a buffer solution, and i
This is because if 1A degree exceeds θ, HA, sufficient sensitivity cannot be obtained.

The complex ion of divalent iron contained in the reagent is not limited in any way, but ferrocyan ion, ferrocene ion, etc. are suitable. However, its concentration must be adjusted correctly, and in the present invention, it needs to be 1 μM or more.

This is the minimum concentration necessary to obtain effective color development.

If necessary, the reagents may be prepared separately into two or more types, but the conditions for each reagent should be adjusted so that each of the above conditions is satisfied when these are mixed to form a single liquid state (state of use). Needless to say, it is necessary. If necessary, other ingredients such as surfactants, preservatives, stabilizers, coexisting substance interference removers (lipase, ascorbic acid oxidase,
(refers to oxidizing agents, etc.) may also be present together. Furthermore, by using a reaction terminator (for example, EDTA, azide, sodium fluoride, etc.), it is also possible to utilize the method of the present invention as an endpoint method.

[Examples] The present invention will be explained in more detail below with reference to Examples, but the following Examples are not intended to limit the present invention.

Example 1 Ceruloplasmin activity in a test solution was measured using the following reagents and the following method.

(Reagent) H2PO4 (PH4,5) ...0.0
5M4-aminoantipyrine (4-AA)...4IQ
MN-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-L toluidine...0.4mM potassium ferricyanide...-0,01% Triton X
-100 (Product name) -0.1% (Measurement method) Add 1% of the above reagent to 50 μm of ceruloplasmin-containing test solution.
, hIL was added and reacted at 37°C, and its absorbance was measured at wavelength 5.
The color development rate was determined by measurement at 50 nm. The reaction curve thus determined is shown in FIG. 1, and the dilution linearity is shown in FIG. 2, respectively.

As is clear from the results shown in FIGS. 1 and 2, ceruloplasmin could be rate assayed in a short time and easily according to the method of the present invention.

Example 2 Using the various reagents shown in Example 1 and setting various composition conditions as shown in Table 1 below, ceruloplasmin activity in the test liquid was measured by the same measurement method as in Example 1.

The measurement results are shown in Table 2 below.

Table 1 Table 2 As is clear from the results in Table 2 above, it is clear that Composition 5, which satisfies all the requirements defined by the present invention, is extremely excellent as a reagent for use in rate assays.

Example 3 Ceruloplasmin activity in a test solution was measured by the following method using the following reagents.

(Reagent) K 82 P Oa -K2 HP 04 (pH 5,
5)・-0,05M4-aminoantipyrine
・"4 mM 3-hydroxy-2,4,6-triiodobenzoic acid...0.5a+
Potassium M ferricyanide -0.02% Triton X -100 (trade name) -0.1% (
Measurement method) Add the above reagent to 50 μl of ceruloplasmin-containing test solution.
Add 0 mft, react at 37°C, and adjust its absorbance to wavelength 5.
The color development rate was determined by measurement at 10 nm. The dilution linearity thus obtained is shown in FIG.

Example 4 Ceruloplasmin activity in a test solution was measured using the following reagents and the following method.

(Reagent) KH2P 04-2 HP 04 (pH 5,5)...
, 0.05M 3-methyl-2-benzothiazolinone hydrazone -0.5mM
N-ethyl-N-(3-sulfopropyl)-m-anisidine...0.4mM potassium ferricyanide...0.02% Triton x-to
o (Product name) -0, 1% (Measurement method) Add the above reagent 1. to 50 μm of the test solution containing ceruloplasmin. O
Add mJZ, react at 37°C, and measure the absorbance at wavelength 59.
The color development rate was determined by measurement at 0 nm.

The dilution linearity thus obtained is shown in FIG.

[Effects of the Invention] As is clear from the above results, according to the method of the present invention, ceruloplasmin activity could be measured quickly and accurately.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the reaction curve of ceruloplasmin in Example 1, FIG. 2 is a graph showing dilution linearity in Example 1, FIG. 3 is a graph showing dilution linearity in Example 3, and FIG. 4 is a graph showing dilution linearity in Example 3. is a graph showing dilution linearity in Example 4. Figure 1 Reaction time (minutes) Dilution series

Claims (1)

[Claims] Ceruloplasmin is produced using any system selected from the group consisting of 4-aminoantipyrine/aniline derivative system, 4-aminoantipyrine/phenol derivative system, and benzothiazolinone hydrazone derivative/aniline derivative system. When measuring activity, pH: 4.0-6
．． 0 Buffer concentration: 0.02M to 0.2M, 4-aminoantipyrine concentration: 1 to 20mM, benzothiazolinone hydrazone derivative concentration: 0.1 to 2mM Reaction system adjusted to satisfy the following conditions. A method for measuring ceruloplasmin activity, comprising measuring ceruloplasmin in a state in which 1 μM or more of divalent iron complex ions are contained.